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Protist, Vol. 167, 106–120, April 2016http://www.elsevier.de/protisPublished online date 3 February 2016

ORIGINAL PAPER

Defining Planktonic Protist FunctionalGroups on Mechanisms for Energy andNutrient Acquisition: Incorporation ofDiverse Mixotrophic Strategies

Aditee Mitraa,1, Kevin J. Flynna, Urban Tillmannb, John A. Ravenc, David Carond,Diane K. Stoeckere, Fabrice Notf, Per J. Hanseng, Gustaaf Hallegraeffh,Robert Sandersi, Susanne Wilkenj, George McManusk, Mathew Johnsonl,Paraskevi Pittam, Selina Vågen, Terje Bergeg, Albert Calbeto, Frede Thingstadn,Hae Jin Jeongp, JoAnn Burkholderq, Patricia M. Gliberte, Edna Granélir, andVeronica Lundgrens

aCollege of Science, Swansea University, Swansea SA2 8PP, United KingdombAlfred Wegener Institute, Am Handelshafen 12, D-27570 Bremerhaven, GermanycDivision of Plant Sciences, University of Dundee at the James Hutton Institute,Invergowrie, Dundee DD2 5DA, United Kingdom (permanent address) and PlantFunctional Biology and Climate Change Cluster, University of Technology Sydney,Ultimo, NSW 2007, Australia

dDepartment of Biological Sciences, University of Southern California,3616 Trousdale Parkway, Los Angeles, CA 90089-0371

eUniversity of Maryland Center for Environmental Science, Horn Point Laboratory,P.O. Box 775, Cambridge MD 21613, USA

fSorbonne Universités, Université Pierre et Marie Curie - Paris 06, UMR 7144,Station Biologique de Roscoff, CS90074, 29688 Roscoff Cedex, France and also CNRS,UMR 7144, Laboratoire Adaptation et Diversité en Milieu Marin, Place Georges Teissier,CS90074, 29688 Roscoff cedex, France

gCentre for Ocean Life, Marine Biological Section, University of Copenhagen,

Strandpromenaden 5, DK-3000 Helsingør, Denmark

hInstitute for Marine and Antarctic Studies, University of Tasmania, Private Bag 129,Hobart, Tasmania 7001, Australia

iDepartment of Biology, Temple University, Philadelphia PA 19122 USAjMonterey Bay Aquarium Research Institute, Moss Landing, CA 95039, USAkMarine Sciences, University of Connecticut, 1080 Shennecossett Rd, Groton CT USA06340

lBiology Department, Woods Hole Oceanographic Institution, Woods Hole MA 02543 USAmInstitute of Oceanography, Hellenic Centre for Marine Research, P.O. Box 2214,

71003 Heraklion, Crete, GreecenDepartment of Biology and Hjort Centre for Marine Ecosystem Dynamics,University of Bergen, P.O. Box 7803, 5020 Bergen, Norway

Corresponding author;-mail A.Mitra@swansea.ac.uk (A. Mitra).

http://dx.doi.org/10.1016/j.protis.2016.01.0031434-4610/© 2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY license(http://creativecommons.org/licenses/by/4.0/).

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Defining Planktonic Protist Functional Groups 107

Institut de Ciències del Mar, CSIC, Ps. Marítim de la Barceloneta, 37-49,08003 Barcelona, SpainSchool of Earth and Environmental Sciences, College of Natural Sciences,Seoul National University, Seoul 151-747, Republic of KoreaCenter for Applied Aquatic Ecology, North Carolina State University, Raleigh,NC 27606 USAAquatic Ecology, Biology Institute, Lund University, Box 118, 22100 Lund, Sweden,and also at Florida Gulf Coast University, Kapnick Center, Naples, Florida 34112 USADepartment of Biology and Environmental Sciences, Centre for Ecology and Evolution in Microbial ModelSystems, Linnaeus University, SE-39231 Kalmar, Sweden

ubmitted March 23, 2015; Accepted January 19, 2016onitoring Editor: Ulrich Sommer

rranging organisms into functional groups aids ecological research by grouping organisms (irrespec-ive of phylogenetic origin) that interact with environmental factors in similar ways. Planktonic protistsraditionally have been split between photoautotrophic “phytoplankton” and phagotrophic “microzoo-lankton”. However, there is a growing recognition of the importance of mixotrophy in euphotic aquaticystems, where many protists often combine photoautotrophic and phagotrophic modes of nutrition.uch organisms do not align with the traditional dichotomy of phytoplankton and microzooplankton.o reflect this understanding, we propose a new functional grouping of planktonic protists in an eco-hysiological context: (i) phagoheterotrophs lacking phototrophic capacity, (ii) photoautotrophs lackinghagotrophic capacity, (iii) constitutive mixotrophs (CMs) as phagotrophs with an inherent capacity forhototrophy, and (iv) non-constitutive mixotrophs (NCMs) that acquire their phototrophic capacity by

ngesting specific (SNCM) or general non-specific (GNCM) prey. For the first time, we incorporate theseunctional groups within a foodweb structure and show, using model outputs, that there is scope forignificant changes in trophic dynamics depending on the protist functional type description. Accord-

ngly, to better reflect the role of mixotrophy, we recommend that as important tools for explanatory andredictive research, aquatic food-web and biogeochemical models need to redefine the protist groupsithin their frameworks.

2016 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BYicense (http://creativecommons.org/licenses/by/4.0/).

ey words: Plankton functional types (PFTs); phagotroph; phototroph; mixotroph; phytoplankton; microzoo-

lankton.

unctional Types in Ecology

n ecology, organism functional categories areften more useful than taxonomic groupingsecause they can be based on ecological func-ion, rather than evolutionary history. Functionalroup descriptions are commonly used by scien-ists to partition the numerous taxonomic classesnto categories more relevant to ecology. Theoncept provides “a non-phylogenetic classifica-ion leading to a grouping of organisms thatespond in a similar way to a syndrome ofnvironmental factors” (Gitay and Noble 1997).unctional group (also referred to as “functional

ype”) classifications thus aid our understandingf ecological processes with applications fromeldwork through to conceptual and mathematicaltudies.

The functional group approach has beenembraced by researchers working on differentorganisms across biomes. Especially when appliedto microorganisms, linking an ecological functionto specific members of a community is oftenchallenging, as individual contributions to rate pro-cesses are difficult if not impossible to measure insitu. Supplementing the classical plant/animal typedichotomy, one of the earliest categorizations ofplankton groups was based on size, driven by prac-tical approaches to plankton sampling (Lohmann1911; Schütt 1892), as well as conforming to typ-ical predator-prey allometries of 10:1 (Azam et al.1983). Various later freshwater and marine stud-ies used such allometric classifications (Sieburthet al. 1978), specifically focussing on phytoplank-ton species (Aiken et al. 2007; Reynolds et al.2002). Kruk et al. (2010) found easily identifiable

108 A. Mitra et al.

morphological differences among phytoplankton tocorrelate with functional properties and proposedsix functional groups; these were based on volume,maximum linear dimension, surface area, and thepresence of mucilage, flagella, gas vesicles, hete-rocytes or siliceous exoskeletal structures. Furtherto morphological characteristics, Weithoff (2003)used resource acquisition capabilities, such asphagotrophy (bacterivory), nitrogen fixation, andsilica usage, to divide phytoplankton into six func-tional groups.

In the context of conceptual and mathemati-cal studies of marine ecology, protist functionalgroups are typically divided simply into “phyto-plankton” (phototrophs) and “microzooplankton”(phagotrophs); the former typically include pho-toautotrophs while the latter represent phago-heterotrophs (e.g., Baretta et al. 1995; Fashamet al. 1990; Plagányi 2007). However, there isnow an increasing recognition that many “phyto-plankton” and photic-zone “microzooplankton” are,in fact, mixotrophic. A wide range of phytoplank-ton ingest prey while various microzooplanktonretain chloroplasts from their prey or harbour pho-tosynthetic endosymbionts and thus contribute toprimary production. Furthermore, many species,when engaging in mixotrophic activity, can attainfaster growth than when in photoautotrophic orphagoheterotrophic mode alone (e.g., Adolf et al.2006; Burkholder et al. 2008; Glibert et al. 2009;Jeong et al. 2010). Thus mixotrophy, defined hereas phototrophy plus phagotrophy, is an inherentcapability of many planktonic protists rather thanbeing the exception that it was previously consid-ered to be (see reviews by Flynn et al. 2013 andreferences therein; Stoecker et al. 2009; Jeonget al. 2010).

Mixotrophic protists are ubiquitous, and com-monly have been found to be dominant infreshwater as well as marine systems from the trop-ics to the poles (e.g., Jeong et al. 2010; Sanders1991; Stoecker et al. 2009; Zubkov and Tarran2008). Yet, most plankton functional type classifica-tions make minimal reference to these mixotrophs.Pratt and Cairns (1985), in their protist-centric func-tional groupings, emphasised strategies to acquireresources. They, thus, divided protists into sixfunctional groups – (1) photo-autotrophic primaryproducers with no distinction made between thosewhich can employ a level of heterotrophy, (2) bacti-and detritivores feeding on bacteria and/or detri-tus, (3) saprotrophs consuming dissolved material,(4) algivores primarily feeding on algae, (5) nonse-lective omnivores feeding non-selectively on algae,detritus and bacteria, and, (6) raptorial predators

feeding primarily on other protozoa and orga-nisms from the higher trophic levels. There is noexplicit mention of mixotrophy. In contrast, Jones(1997) and Stoecker (1998) specifically focussedon how groups of protists combine phototrophyand phagotrophy to support growth (Fig. 1). Jones(1997) primarily focussed on the mixotrophic flagel-lates, proposing four functional groups according totheir photosynthetic and heterotrophic capabilities.Stoecker’s (1998) classification included a widergroup of protists. In addition to flagellates, Stoecker(1998) accounted for ciliates, sarcodines and pro-tists with algal symbionts – groups which had notbeen included in the studies of Pratt and Cairns(1985) and Jones (1997).

It is now clear that the time has come to abandonthe premise that protists are either “little plants” or“little animals”, to move away from the misplaceddichotomy of “phytoplankton” versus “microzoo-plankton” (Flynn et al. 2013). How can we bestreclassify them for ecological studies? Dependingon the character of interest, there is scope to createmany types of functional groups. For the applica-tion of functional classifications to mathematicalmodels, however, a simpler approach is requiredin order to constrain computational loads while pri-oritising functionality to biogeochemistry and/or totrophic (food web) dynamics. Based on our under-standing of aquatic ecology, coupled with improvedunderstanding of how protists have evolved, wesuggest that a reappraisal is required of the defini-tion of functional group descriptions for planktonicprotists, now explicitly including mixotrophs. Wepropose a new, ecologically based, functional groupclassification for aquatic planktonic protists. This isthe first time that all the three groups of planktonicprotists – phytoplankton, mixotrophs and microzoo-plankton – have been considered explicitly underthe functional group approach. The group divi-sions we propose are based on both energy andnutrient acquisitions, and are consistent with themain drivers for conceptual and mathematical mod-elling (i.e., biogeochemistry and trophic dynamics).We discuss the importance of these groups asdescriptors for future research on planktonic protistcommunities.

Classifying Protist Functional Groups

In evolutionary terms, heterotrophy (originallyosmotrophic or saprotrophic, later includingphagotrophy) is the ancestral state in protistswhile photo-autotrophy is the derived and morerecent state (Raven 1997; Raven et al. 2009). The

Defining Planktonic Protist Functional Groups 109

TYPE I“Idea l” mi xotroph ; Balanced pho totroph y an d phago troph y

TYPE IIPhago troph ic “algae ”; Primarily Pho totrophic

TYPE IIAFeed when DIN is limiting

e.g., Prorocen trum mi nim um

TYPE IIBFeed unde r trace organ ics growth

factor limitatio ne.g., Ur oglena american a

TYPE IICFeed unde r ligh t limit atio n

e.g., Ch rysoch romulin a brevifilu m

GROUP BPrimarily pho totrophic;

feed unde r ligh t limit atio ne.g., Ch rysoch romulin a

brevifilu m

GROUP CPrimarily pho totroph ic; feed for essentia l eleme nt (e.g., iron ) or

growth sub stance (e.g., phospholipids )

e.g., Ur oglena american a GROUP DPrimarily pho totrophic; very low inges tion rate; inges tion of prey and/or up take of organ ics to aid cell maintenance unde r se vere

ligh t limit ation e.g., Cr yptomona s ovata

TYPE IIIPho tosynthetic “protozoa ”; Prima rily Phago trophic

TYPE IIIAC-fix unde r prey limi tation ; ha ve plastid s

& assimi lates DINe.g., Poteriooch romona s malhamensis

TYPE IIIBProtozoa with acqu ired pho totroph y through

harbou ring endo symbionts , plastid seques tration C-fix to supp lement C nu trition

e.g., plastid retaining Laboe a strob ilia ,symbiont ha rbou ring green No ctiluca scintillan sGROUP A

Growth dependen t on he terotrophy; phototroph y supp lements growth rate

e.g., Poteriooch romona s malhamensis

Figure 1. Traditional classification of mixotrophic protists according to Stoecker (1998; open boxes) and Jones(1997; grey boxes). “Groups” proposed by Jones (1997) have been aligned against “Types” proposed byStoecker (1998). DIN, dissolved inorganic nutrients.

developmental lines from phago-heterotrophy tophoto-autotrophy have occurred through a seriesof evolutionary pathways with gains and lossesof physiological functionality (Raven et al. 2009).Notably, there have been sequences of acquisitionand then loss of the capability to photosynthesize(e.g., Delwiche 1999; Saldarriaga et al. 2001;Van Doorn and Yoshimoto 2010; Wisecaver andHackett 2010). That capability, and the natureof its expression in extant protists, is of suchfundamental importance that it usefully forms thebasis of a functional group classification.

Broadly following the direction of protist evolu-tionary pathways, we propose a division of protistsaccording to the schematic shown in Figure 2;

differences in physiological processes betweenthe different groups are highlighted in Figure 3. Allprotists appear to be osmotrophic to some degree,if only for certain vitamins and to re-acquire leakedprimary metabolites such as protein amino acids(Flynn and Berry 1999). Accordingly, we do not useosmotrophy as a functional group characteristic. Incontrast, the presence or absence of phagotrophyand/or of phototrophy are clear defining char-acteristics that have profound consequences forbiogeochemistry and trophic dynamics throughthe operation of predator-prey interactions (Flynnet al. 2013; Mitra et al. 2014a). In what follows, wethus place emphasis on photo- and phago- trophyas classifying processes.

110 A. Mitra et al.

START WITH PROTIST

Capab le of carbon (C ) fixation ?

YES NO

Phago-heterotrophs/ Osmotroph se.g., Oxyr rhis ,

Fig. 3(A)

Phagoc ytose?

Photo-autotrophse.g., diatoms

Fig. 3(B)

Constitutive/inna te capability forC-fixation ?

Con stitutive Mixotroph s

e.g., Ka rlod inium , Pry mnesi um

CM; Fig . 3(C)C- fixation capabilities

acquired from specific prey?

GeneralistNon-Co nstitutive

Mixotroph se.g., Labo ea strobila, Str ombidi um capit atum

GNCM; Fig. 3(D)

C-fixation mediated by symbionts?

plastidicSpecialist

Non-Co nstitutive Mixotrophs

e.g., Mesodini um, Dinoph ysis

pSNCM ; Fig . 3(E)

endosymbioticSpecialist

Non-Co nstitutive Mixotroph s

e.g., Collo zou m, gre en Noc tiluca

eSNCM; Fig. 3(F)

NO

YES

YES

NO

NO

YES

NO

YES

Figure 2. Flowchart showing the pathways used to derive the functional groups we propose to classify theplanktonic protists. See also Figure 3.

We identify functional groups at the extremeends of the spectrum as (i) phagotrophs, whichconform to the common concept of “microzoo-plankton” (including heterotrophic nano-flagellates;Figs 2 and 3A), and, (ii) phototrophs incapable ofphagocytosis that conform to the common conceptof “phytoplankton” (Figs 2 and 3B). The mixotrophicprotists, combining phago- and photo- trophy in asingle cell, are first divided with respect to phototro-phy between constitutive (inherent or innate) versusnon-constitutive (acquired) capabilities. Constitu-tive mixotrophs (CMs; Figs 2 and 3C) have theinnate ability to photosynthesize – that is, theyhave vertical transmission of plastids and, pre-sumably, the ability to regulate plastid function viaprotist nuclear-encoded genes. Non-constitutivemixotrophs (NCMs, Fig. 2), in contrast, acquire thecapability to photosynthesize from consumption ofphototrophic prey. They depend on horizontal trans-mission of plastids or symbionts. The NCMs canthen be divided into generalists and specialists.Generalist non-constitutive mixotrophs (GNCMs;Figs 2 and 3D) can use photosystems sequesteredfrom a broad range of phototrophic prey. Specialistnon-constitutive mixotrophs (SNCMs; Fig. 2) havedeveloped a need to acquire the capacity for photo-synthesis from one or a few specific sources. This

SNCM grouping can then be further divided intothose which are plastidic (pSNCMs, Figs 2 and 3E)and those which contain endosymbionts (eSNCMs,Figs 2 and 3F).

Constitutive mixotrophs (CMs, Fig. 3C) conformto the common perception of mixotrophic protists asunicellular algae that can consume other organisms(Sanders and Porter 1988). The CM group includesrepresentatives from a wide range of eukaryotic“phytoplankton” (almost all major phototrophic pro-tist groups excluding diatoms; Flynn et al. 2013;Jeong et al. 2010), ingesting various prokaryotic(e.g., cyanobacteria, bacteria) and eukaryotic prey(e.g., ciliates, dinoflagellates, cryptophytes, amoe-bae; Burkholder et al. 2008; Jeong et al. 2010;Stoecker et al. 2006; Tillmann 1998; Zubkov andTarran 2008).

Non-constitutive mixotrophs (NCMs, Fig. 3D-F) lack an inherent (constitutive) ability to fullysynthesize, repair and control the photosyntheticmachinery (Flynn and Hansen 2013). Mecha-nisms differ among representatives, but they allengulf photosynthetic prey. They may then retainthe prey as symbionts through a process termedendosymbiosis. Alternatively, they retain parts ofthe ingested prey necessary for photosynthe-sis – chloroplasts (kleptoplastidy), along with,

Defining Planktonic Protist Functional Groups 111

(A) Phago-heterotroph (B) Pho to-au totroph (C ) Con s�tu�ve Mixotroph ; CM

Growth

Phagotrophy

DIMDOM

Growth

Phototrophy

DIM

DOM

CO2

Growth

Phagotrophy

Phototroph y

DIMDOM

CO2

Growth

Phagotroph y

Phototroph y

DIM

DOMCO2

(D) Generali st Non-Con s�tu�ve Mixotroph; GNCM

(E) plas�dic Special istNon-Con s�tu�ve Mixotroph; pSNCM

Growth

Phagotrophy

DIM

DOM

Phototrophy

CO2

(F) end osym bio�c Sp eciali stNon-Con s�tu�ve Mi xotro ph; eSNCM

Growth

Phagotrophy

Photot rop hy

DOM

CO2

DIM

Figure 3. Schematic illustrating the different levels in complexity among different types of protist. (A)phagotrophic (no phototrophy); (B) phototrophic (no phagotrophy); (C) constitutive mixotroph, with innatecapacity for phototrophy; (D) generalist non-constitutive mixotroph acquiring photosystems from different pho-totrophic prey; (E) specialist non-constitutive mixotroph acquiring plastids from a specific prey type; (F) specialistnon-constitutive mixotroph acquiring photosystems from endosymbionts. DIM, dissolved inorganic material(ammonium, phosphate etc.). DOM, dissolved organic material. See also Figure 2.

sometimes, the prey nucleus (karyoklepty) andmitochondria, making use of these for a period oftime (see reviews by Johnson 2011a, b and refer-ences therein; Stoecker et al. 2009). The retentiontime (for kleptoplastids) varies from hours to daysor longer, depending on the mixotroph and the prey.

The GNCM group (Fig. 3D) uses chloroplastsderived from several to many prey types (e.g.,Laval-Peuto and Febvre 1986; Laval-Peuto et al.1986; Schoener and McManus 2012; Stoeckeret al. 1988, 1989). About a third of the ciliates(by numeric abundance) inhabiting the marinephotic zone fall within this GNCM functional group(Blackbourn et al. 1973; Calbet et al. 2012; Dolanand Pérez 2000; Jonsson 1987; Laval-Peuto andRassoulzadegan 1988; McManus et al. 2004; Pittaand Giannakourou 2000; Pitta et al. 2001; Stoeckeret al. 1987). The ability to maintain an acquiredphotosynthetic capacity by GNCMs is poor, andfrequent re-acquisition is required.

In contrast to the GNCM group, SNCMs acquirephotosystems from only specific prey. Special-ization ranges from harbouring only plastids toharbouring intact cells (protists or cyanobacte-ria) as symbionts. Maintenance of the acquiredphotosystems is usually good, so that SNCMscan modulate photosynthesis (photoacclimate andundertake damage repair) similar to that seen inCMs but absent in GNCMs. Among the SNCMs, thepSNCM sub-group (Fig. 3E) includes ciliates suchas Mesodinium rubrum (Garcia-Cuetos et al. 2012),which feed on several prey types, but acquire pho-tosynthetic apparati (and nuclei) only from specificcryptophyte clades (Hansen et al. 2012; Johnson2011a, b; Johnson et al. 2007), the dinoflagel-late Dinophysis, which sequesters plastids fromthe ciliate M. rubrum (Park et al. 2006), and,an undescribed Karlodinium-like dinoflagellate thatacquires plastids from the haptophyte Phaeocystisantarctica (Gast et al. 2007; Sellers et al. 2014).

112 A. Mitra et al.

The photosymbiotic eSNCM sub-group (Fig. 3F)includes, within marine systems, the biogeochemi-cally important and cosmopolitan Foraminifera andRadiolaria (Acantharia and Polycystinea). Thesemixotrophs harbour and maintain dinoflagellate,haptophyte, or green algal endosymbionts. Theendosymbionts are acquired during the juvenilestages and maintained throughout most of the lifecycle (Caron et al. 1995). The presence of theseendosymbionts is obligatory for normal growthand reproduction in eSNCMs (Caron et al. 1995;Decelle et al. 2012; Langer 2008; Probert et al.2014). Freshwater ciliates (Paramecium bursaria,Vorticella spp, Stentor spp, Frontonia spp, Stoke-sia spp and Euplotes spp) specifically harbour themicroalga Chlorella as endosymbionts (Berningeret al. 1986) and thus falls within the eSNCM cate-gory. However, in contrast to the marine eSNCMs,the freshwater mixotrophs do not require the sym-bionts for reproduction (Dolan 1992).

Proposed versus Other FunctionalGroup Classifications

Our proposed grouping strikes at the very basisof ecophysiology - whether an organism is a pri-mary producer, a consumer, or some combinationof the two (mixotrophic). These are key featuresaffecting contributions to biogeochemistry and/ortrophic interactions. We now compare our proposalto earlier classifications of protists that consideredmixotrophy.

Comparison of the mixotrophic functional groupsof Jones (1997) to our proposed grouping (Fig. 1versus Fig. 2) reveals that all the groups proposedby Jones are constitutive mixotrophs (CM, Fig. 3C)because they have an innate capability to photo-synthesize. In contrast, the groupings by Stoecker(1998) include both constitutive (Stoecker’s TypesIIA, B, C and IIIA in Fig. 1; CM, Fig. 3C) andnon-constitutive mixotrophs (Stoecker’s Type IIIBin Fig. 1; NCM, Fig. 3D-F). The Types IIA, B andC of Stoecker (1998, Fig. 1) could thus in essencebe phytoflagellates within the constitutive functionalgroup.

The prime discriminators for the functional groupdescriptions of Jones (1997) and Stoecker (1998)are the balancing of energy and nutrient supply anddemand. Thus, groups were split according to theproportion of phototrophy versus phagotrophy, withthe lesser activity “topping up” the least abundantresource (carbon, nitrogen, phosphorus, iron etc.).However, now we view mixotrophy as likely per-forming a synergistic rather than a complementary

role in nutrition (Adolf et al. 2006; Wilken et al.2014a; and as modelled by Flynn and Mitra 2009).Repression and de-repression across the range ofnutrient and energy acquisition options modulateexpression of phototrophic versus phagotrophicactivities. There is great variation across theconstitutive mixotrophs (CM group) in this regard,and also in growth rate potential. For example, ithas been shown that when conditions are optimalfor mixotrophy (i.e., sufficient light and prey areavailable), some dinoflagellates have a highergrowth rate compared to their growth when func-tioning as phototrophs (low or no prey available)or phagotrophs (under light limitation) (Jeong et al.2010). The conditions specified by Jones (1997)and Stoecker (1998) could, therefore, be viewed asa secondary level of classification to that we nowpropose, describing placement of mixotrophs uponsliding scales of phototrophy versus phagotrophy,depending on their physiological capabilities andresource availabilities.

We can thus envision a series of functional groupdescriptions ranging from the potential to engagemixotrophy (our proposal), to expression of mixotro-phy according to physiological stressors (cf. Jones1997; Stoecker 1998), to utilizing carbon dioxideor particulate food (bacteria/ detritus, algae, het-erotrophic protists and animals) as a source ofcarbon (Pratt and Cairns 1985). The first division,however, must be between protist groups that arenon-phagotrophic phototrophs, phagotrophs withno phototrophic capacity, or mixotrophs with somecombination of the two (i.e., between the CM,GNCM and SNCM groups).

Ecological Implications – aDemonstration

The purpose in grouping organisms according tofunctionality is to aid our understanding of ecology.Arguably, the most fundamental ecological divisionis between primary producers and their consumers.Interactions between these two groups and highertrophic levels form cornerstone components in eco-logical research and modelling (e.g., Cohen et al.1993). The mixotrophic protists combine facets ofboth primary producer and consumer in one organ-ism. The ability to express these facets allows, andmay actually require, us to divide them into CM,GNCM and SNCM functional groups (Figs 2 and 3).Each of these groups display different interactionsand dynamics with other plankton. As an example,we show here the contrasting behaviour of a sim-ple trophic food web, in which a particular protist is

Defining Planktonic Protist Functional Groups 113

DIM

DOM

Phytoplankton

μZ

Bacteria

(A)

DIM

DOMBacteria

(B)

Phytoplan kton

GNCM

DIM

DOMBacteria

(C)

CM

Phytoplan kton

Figure 4. Three contrasting simple food-web struc-tures. Scenario A portrays the classic paradigm wherephytoplankton and microzooplankton are the two pro-tist plankton functional types. Within Scenarios B andC, the microzooplankton functional type is replacedby the non-constitutive mixotrophs (NCMs) and con-stitutive mixotrophs (CMs) respectively. The releaseand uptake of dissolved inorganic matter (DIM, ammo-nium, phosphate etc.) is indicated.

operating as a strict phagotroph (“microzooplank-ton”), as a GNCM, or as a CM.

Method – the foodweb model framework. Todemonstrate the potential impact of the differentprotist functional groups on trophic dynamics, wecompare the outputs from three contrasting insilico plankton foodweb structures operating in amesotrophic setting:

(i) Scenario A: traditional foodweb structure(Fig. 4A). This framework includes photo-autotrophic (non-phagotrophic) protist

phytoplankton as primary producers, thephago-heterotrophic microzooplankton (�Z)which graze on these phytoplankton andbacteria as decomposers.

(ii) Scenario B: an alternative food web frame-work incorporating GNCMs (Fig. 4B). Thisfood web structure includes the same com-ponents as Scenario A, except that the�Z are replaced with GNCMs. The GNCMsdemonstrate acquired phototrophy throughsequestration of the photosynthetic apparatusfrom the phytoplankton prey.

(iii) Scenario C: the third food web frameworkincorporates CMs (Fig. 4C). Again, the foodweb structure includes the same componentsas Scenario A, except that the �Z are replacedwith CMs. The CMs photosynthesize usingtheir constitutive chloroplasts and attain addi-tional nutrition (C,N,P) through the ingestion ofthe phytoplankton prey.

The food web model structure for the three sce-narios was adapted from Flynn and Mitra (2009).In brief, this consists of a mixed layer depth of25 m, inorganic N of 5 �M, and inorganic P of0.625 �M, with an effective cross mixing-layer dilu-tion rate of 0.05 d-1. The model includes variablestoichiometric (C:N:P) acclimative descriptions ofthe plankton. When we first explored modelling theecophysiology of mixotrophic protists, we found thatwe had to split the potential for mixotrophy intogroupings of what we now term here as CMs andNCMs. The “perfect beast” model of Flynn andMitra (2009) contained switch functions that couldconfigure between these types, together with accli-mation descriptions to enable them to represent allthe functional groups described by Jones (1997)and Stoecker (1998). This model is used here. Forfurther details of the description of the model config-urations, please see the Supplementary Material.

Results from in silico experiments. The tem-poral and spatial development of biomass of thedifferent functional groups within the simulatedcommunities are very different under the three sce-narios (Fig. 5A-C); additional plots are presentedin Supplementary Material Figures S1-S3. In sce-nario A, the dynamics follow those expected froma typical predator-prey system. However, in bothscenarios B and C, the mixotroph functional groupsoutcompete the phototrophic phytoplankton (here-after, phytoplankton). Indeed, in scenario C, theCMs ultimately become the dominant functionalgroup (akin to a bloom situation). In scenario B,in contrast, the GNCMs could only attain a limitedproductivity due to their dependency upon the

114 A. Mitra et al.

Figure 5. Temporal pattern of the development ofthe biomass in the simulated communities underthe three scenarios. Also shown are the cumulativeprimary productivities by phytoplankton (phyto) andmixotroph (mixo) over the 30-day simulation periodunder the three alternative scenarios. No mixotrophsare present in Scenario A. See also Figure 4.

phytoplankton for the supply of plastids to allowphotosynthesis.

The implementation of a GNCM versus a CMmixotroph thus generates an interesting dynamicto their phototrophic ecology. The GNCMs cannever dominate the system. Due to their depend-ency on prey to acquire phototrophic capabilities(Figs 2 and 3D), GNCM blooms would always ter-minate through exhaustion of prey (SupplementaryMaterial Fig. S2). CMs, however, through a com-bination of phagotrophy and phototrophy have theadvantage and the capability to dominate a sys-tem, ultimately forming successful blooms. Indeed,harmful algal bloom species are typically CMs(Burkholder et al. 2008). In essence, CMs act asintraguild predators, both feeding on and competingwith their prey; thus in contrast to specialist preda-tors (i.e., NCMs) dependent on specific prey items,CMs can suppress their prey much more strongly(Wilken et al. 2014b). For CMs and for phytoplank-ton, self-shading resulting in light limitation maybecome of consequence for primary productivity;for the final days of production in a GNCM system,this light limitation is of lesser importance as the pig-ment content of the water column degrades rapidlyduring the final stages of the bloom (see Flynn andHansen 2013).

Comparison of the cumulative primary produc-tivity, by phytoplankton and the mixotrophs (phytoand mixo, respectively, bottom panel in Fig. 5) overthe 30-day simulation period under the three alter-native scenarios, showed a substantially higheramount of primary production when CMs wereimplemented (Fig. 5; C-fix in Supplementary Mate-rial Figs S1-S3). Production of dissolved organicsoriginating directly from primary production wassimilarly enhanced for CMs (Supplementary Mate-rial Fig. S3; cf. Figs S1 and S2). The regenerationof dissolved inorganics, typically associated withpredatory activities, was enhanced where GNCMswere implemented (scenario B; SupplementaryMaterial Fig. S2; cf. Figs S1 and S3).

As yet we know little detail about the mechanismsused by mixotrophs to modulate their photoauto- vsphagohetero- trophic capabilities. Different speciesmay occupy different regions of the continuum froma phototrophic extreme to a phagotrophic extreme,while the ecophysiology of others will be predom-inantly photoauto- or predominantly phagohetero-trophic. The critical issue, then, is whether the pro-tists are dependent upon other organisms (i.e.,NCMs, Fig. 3D-F) for the acquisition and continua-tion of their mixotrophic potential, or if they possessthe full genetic and/or physiological capacity to

Defining Planktonic Protist Functional Groups 115

undertake both modes of nutrition all of the time(i.e., CMs, Figs 2 and 3C).

Discussion

A major reason for dividing the protists into theproposed functional groups (Figs 2 and 3) isthe recognition of the differences in the con-sequential population dynamics and role of thegroups in ecosystems (Figs 4 and 5). The rolesof the non-phagotrophic and non-phototropic forms(representative of traditional “phytoplankton” and“microzooplankton”) are established. A role of CMsis largely acknowledged in the literature, althoughdiscussions are dominated by alternate energysupply options, while we suggest the roles of photo-and phago- trophy are more likely linked to syn-ergy in energy and nutrient supply routes (see alsoWilken et al. 2014a, b). The CMs have nonethe-less drawn only limited attention of modellers. Thescope for a revision of the ecological role of CMsis illustrated by their suggested relationship withbacteria, wherein especially nano-sized CMs pro-mote bacterial growth by release of DOM, andthereby gain nutrients they would otherwise beunable to acquire (Mitra et al. 2014a). The GNCMgroup is expected to have quite different populationdynamics from other mixotrophs, being dependent(within a generation time) on a repeated inges-tion of phototrophic prey for chloroplasts. Flynnand Hansen (2013) indicate some differences inthese dynamics, but there likely is much more toexplore, related to the effects of mixotrophy onassimilation efficiency and of photon dose on thelongevity of acquired plastids. The SNCMs at firstsight may be considered similar to CMs, but thereare sharp contrasts in the nature of the host (cil-iate, Foraminifera etc.) in comparison with that ofCMs (phytoflagellates, dinoflagellates etc.) and ofthe main prey types. While the need for donors ofphototrophy (as plastids or as endosymbionts) isless frequent for SNCMs than for GNCMs, the spe-cialism in that need places an additional dynamicin their relationship with other planktonic membersof the ecosystem.

The ecology of CMs is in some ways rela-tively simple, as they do not need to acquire theirmixotrophic potential (for phototrophy) from anotherorganism. Nevertheless, there are sound reasonsto sub-divide CMs into those that consume bacteria(Hartmann et al. 2012; Unrein et al. 2014; Zubkovand Tarran 2008) versus those capable of (also)consuming non-bacterial prey (Burkholder et al.2008; Stoecker et al. 2006). This is especially true

if the latter are competitors in terms of phototro-phy, or even potential predators of the mixotrophs(Thingstad et al. 1996). The simulations run here(Fig. 5) were configured for a mesotrophic sys-tem where phagotrophy by �Z, GNCMs and CMspredominantly involves ingestion of phytoplanktonprey. Within the three scenarios (Figs 4 and 5), itwas assumed that the predators consumed onlyphytoplankton. The range of size of the protist phy-toplankton group in these systems could vary byorders of magnitude (e.g., nano- to micro- sized).Likewise, their protist grazers could occupy a largesize spectrum. For example, the size spectrum forthe prey for GNCMs (15-60 �m ESD) can varybetween 1-40 �m ESD (McManus et al. 2012;Stoecker et al. 1987). Divisions between thosecapable of consuming different prey sizes may beachieved via allometric considerations. However,there are plenty of examples of mixotrophs feed-ing on prey larger than themselves and, also, oflarger species feeding on bacteria or picocyanobac-teria (Glibert et al. 2009; Granéli et al. 2012; Jeong2011; Jeong et al. 2005, 2012; Lee et al. 2014;Seong et al. 2006). Accordingly, functional groupdivisions that either partition protists solely accord-ing to predator-prey allometrics (e.g., Sieburth et al.1978), or have a sliding scale for photoauto- ver-sus phagohetero- trophy (e.g., Jones 1997), appearinsufficiently robust from an ecological perspective.Below we consider each group in more detail.

CM group. In the photic zone plankton, CMs(Fig. 3C) often dominate eukaryotic microbialbiomass (cf. Supplementary Material Fig. S3), bothin eutrophic and oligotrophic systems across all cli-mate zones (Burkholder et al. 2008; Hartmann et al.2012; Havskum and Riemann 1996; Li et al. 2000a,b; Sanders and Gast 2012; Stoecker et al. 2006;Unrein et al. 2007, 2014). Mixotrophic phytoflag-ellates have accounted for 50% of the pigmentedbiomass during non-bloom conditions off Den-mark (Havskum and Riemann 1996). Constitutivemixotrophy has been identified as a major modeof nutrition for harmful phytoflagellate species ineutrophic coastal waters (Burkholder et al. 2008;Jeong et al. 2010; Stoecker et al. 2006) and CMscan account for significant and occasionally dom-inant predation pressure on bacteria (Hartmannet al. 2012; Sanders et al. 1989; Unrein et al.2014; Zubkov and Tarran 2008). For example,it has been shown that mixotrophy plays a vitalrole in the trophic dynamics of the oligotrophicgyres (Hartmann et al. 2012; Zubkov and Tarran2008). Modelling this ecosystem using the tradi-tional paradigm (Scenario A, Fig. 4) would fail toportray the correct dynamics and, thus, would be

116 A. Mitra et al.

misleading. Most of the phytoplankton in thesesystems are bacterivores, and without mixotrophy(photoautotrophy plus bacterivory), primary pro-duction would be much lower due to N and Pstarvation.

The differences between CMs and NCMs areclear and unambiguous. While each group includesexamples of the sliding scale and allometrics, wejustify the split based on the definition of thesource of the phototrophic potential (innate versusacquired), because this has profound impacts forthe ecophysiology of the organisms.

GNCM vs SNCM groups. Up to about one-third of photic zone ciliate microzooplankton areGNCMs (Calbet et al. 2012; Dolan and Pérez 2000;McManus et al. 2004; Pitta et al. 2001; Fig. 3D).In summer stratified surface waters, more than50% of ciliates have on occasion been found to bemixotrophic (Calbet et al. 2012; Putt 1990; Stoeckeret al. 1987). The contribution of these GNCMs to“phytoplankton” biomass (as chlorophyll) can thusbe substantial (Stoecker et al. 2013; see also Fig. 5and Supplementary Material Fig. S2). They cancomprise a significant proportion of all zooplank-ton, and their ecology is not only linked to theirmixotrophic capabilities, but also limited by theirneed to consume phototrophic prey in order toacquire chloroplasts.

The SNCMs (Fig. 3D-F), requiring specific prey,present a different ecological dynamic in compari-son with the CMs and GNCMs. They do not needto interact with specific prey often, but when theyneed to do so, they must consume one of only afew prey options in order to acquire phototrophy.That restriction has important implications for theSNCMs and for their specific prey. In blooms, thecosmopolitan SNCMs Mesodinium rubrum and M.major can account for approaching 100% of plank-ton biomass (Crawford et al. 1997; Garcia-Cuetoset al. 2012; Herfort et al. 2012; Montagnes et al.1999; Packard et al. 1978). This is possible as theneed for plastids by the red Mesodinium species isoccasional (Johnson and Stoecker 2005; Johnsonet al. 2006), and acquired cryptophyte plastids caneven replicate within the ciliate (Hansen et al. 2012;Johnson et al. 2007).

The Foraminifera and Radiolaria (Acantharia andPolycystinea) eSNCMs (Fig. 3F) contribute signifi-cantly to primary production and trophic dynamicsin oligotrophic oceanic gyres, and thus play a vitalrole in marine biogeochemistry (Caron et al. 1995;Decelle et al. 2013; Dennett et al. 2002; Gast andCaron 2001; Michaels et al. 1995; Stoecker et al.2009; Swanberg 1983). These eSNCMs, the dom-inant large planktonic predators in the subtropical

gyres, probably could not survive and grow in thisnutritionally dilute environment without the C sup-plement from their symbionts (Caron et al. 1995),suggesting a major role of mixotrophy in shapingthe trophic structure of subtropical gyre ecosys-tems.

Conclusion

Functional group descriptors are specificallyintended to aid our understanding of ecology (Gitayand Noble 1997; Smith et al. 1997). Arguably theultimate test of that understanding comes from anability to construct and use mathematical modelswhich display behaviours that align with expecta-tions gained from empirical knowledge.

Based on experimental and modelling studies,we now realise that mixotrophy in protists can bea synergistic process and does not just provide a“top-up” or “survival” mechanism (Adolf et al. 2006;Mitra and Flynn 2010; Mitra et al. 2014a; Vågeet al. 2013). The nature of that synergism betweenphoto- and hetero- trophy is ultimately modulatedby whether the phototrophic capacity is constitu-tive (innate) or acquired. Accordingly, we proposethat the functional group descriptors for plank-tonic protists should align with the innate capacity,or otherwise, for phototrophy and/or phagotrophy(Fig. 2). A division on these grounds makes sensefor modelling, both from evolutionary and ecolog-ical perspectives, in that these groups are clearand unambiguous. Such traits are important fea-tures in functional group definitions. Within thisdivision, the groups described by Jones (1997) andStoecker (1998) form important secondary func-tional group descriptions, while those by Pratt andCairns (1985) form a tertiary level of description forboth mixotrophic and heterotrophic protists.

Over the past decade there has been a drive tomodify biogeochemical and aquatic food web mod-els through incorporation of the functional groupapproach. Within these models, plankton functionaltypes are increasingly deployed to aid descriptionsof processes from biogeochemistry to fisheries(e.g., end-to-end models; Rose et al. 2010). Theprimary focus has been on splitting the “phyto-plankton” into several groups – for example, into“diatoms” requiring Si, “calcifiers” requiring calcium,etc. – with each group having its own state vari-ables (e.g., Baretta et al. 1995). Far less emphasishas been placed on expanding the “zooplankton”component (see Mitra et al. 2014b and referencestherein), while “mixotrophic” groups are typicallyignored altogether. Modellers typically start with

Defining Planktonic Protist Functional Groups 117

an attempt to simplify the system sufficiently toenable or assist computation and analysis. Thatsimplification process must not be so great thatthe critical linkage to reality is lost. Given ourrenewed appreciation of mixotrophy (Flynn et al.2013 and references therein), we suggest that acomplete overhaul of the core structure of biogeo-chemical and plankton food web models may bewarranted, in order to provide a more accurate eco-logical perspective on ecosystems functioning thatacknowledges the existence of mixotrophs.

Acknowledgements

This work was funded by grants to KJF and AMfrom the Leverhulme Trust (International NetworkGrant F00391 V) and NERC (UK) through its iMAR-NET programme NE/K001345/1. The University ofDundee is a registered Scottish charity, numberSC015096. AM would like to thank Rohan Mitra-Flynn for his patience and co-operation.

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