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Home , Euglenid, Katablepharid, Palpitomonas bilix

Yubuki University SouthBohemia, 37005 of MA 02543,USA

This article is protected bycopyright. reserved. All rights 10.1111/jeu. differences between thisversion and the Version Record. of Please cite thisarticle as doi: through the copyediting, typesetting, pagination for publication accepted This articlehasbeen diversity ofnatural envi within thecontext characterizati astheexploration, defined here remainIn limited. thisreview, we discuss the challenges associated “field with ”, co naturalprotist from obtained data because about thediversity,ecology, hi evolutionary and evolutionary history theoverall for understanding The discoverycharacterization and ofprotist communities fromdiverse environments are crucial ABSTRACT [email protected]@mail.ubc.ca and Telephone 6048224892;FAX +1 number: 8226089;emails: number:+1604 Vancouver, BritishColumbia ResearchMuseum, University British Centre and of Blvd., University Columbia, 6270 T.and N. Heger J. Yubuki, The Departments Correspondence d b a Heger J. Thierry and Evolution of Microbial Diversity, the toBetter Understand A Ecology, Essential Research is Field in Resurgence Protistology Field of Importance --- al. et Heger Article type :Symposium Article Date:29-Oct-2013 Accepted Revised Date:21-Oct-2013 :15-Mar-2013 Date Received c USA University BritishColumbia, of Vancouve

Division ofInvertebrate Zoology, Mu American Institute ofParasitology, Biology Centre, Czech Academy Facultyand ofSciences, ofScience, The Departments Botany and Zoology,of Beat Accepted ArticleWoodsInstitution, Geolog HoleOceanographic a

12095-4379 12095-4379 a

, Virginia P. Edgcomb , V6T Canada 1Z4, Č eské Bud b , EunsooKim r, BritishColumbia, V6T 1Z4, Canada

and undergone full peer review buthas notbeen undergonereview and full peer

mmunities, especially of heterotrophic species, heterotrophic species, of especially mmunities, ě and proofreading process, which may lead to on, andinterpretationof ronments orfieldexperiments, andprovide seum ofNaturalHistory,seum New York, NY, 10024, jovice, Czech Republic Czech jovice, y Biodiversity Research of Botany andZoology, Beaty Biodiversity story of remain unanswered, notably notably unanswered, story protistsremain of y Department, andGeophysics Woods Hole, of life on earth. However, earth. lifeon questions of major c , Julius Lukeš , Julius d , Brian S.Leander

microbial eukaryotic microbialeukaryotic Centre and Museum, Centreand a & Naoji Despite numerous field studies since the17 studies since field Despite numerous Protists are understudied toplants, incomparison The importance protistan of fieldwork of understanding significantly improvedour limited forprotists,we fully recognize that th environments orfieldexperiment characterization and interpretation of microbial eukaryotic diversitywithin the context ofnatural future offieldprotistology. define we Here, attention draw and toseveraltend (3) protists, tobelimited challenges associated for withthe important insights intofundament the field are limited. The extent ofourignorance limited. are The extent the field evolutionary theseorganisms history of remain of thebi eukaryotic component

This article is protected bycopyright. reserved.All rights during the20 uponbyexpanded “fieldprotistologists” numerous particularlyfrom oxygen-depleted environments. This rich legacyin protistology has been ot andmany (1877-1946) Alfred Kahl and soilhab from freshwater amoebae species Brady workedons who (1835-1891) first classifications produced the asprotists firstrecognized were beau and detailed incredibly others contributed ErnstHaeck (1832-1871), Edouard Claparède that andduring microscope lensesoccurred dedicated toprotists, Animalcu the uniquenessoforganisms hewasobserving 1 Lechevalier 1923; microscopic protists(Dobell microscopy light advanced Müller (1730-1784) by work pioneering Leeuwenhoeck van Antoni 1600’sFIELD the studiessince fo foundation formthe ; . Keywords climate. achanging to adaptations ecosystem larger in roles andtheir functions, of novelevolutionary patte ex that lineages ofnew discovery the (1) toward combinemol field studiesthat suggestions tohelp fill this important gap inknowledge. We also argue that increased efforts in AcceptedLeidyEugène Pe (1823–1891)and crui in 1872,thefirstscientific Article The purposeofthisreviewisto(1)highlig foodwebs fieldresearch; culture; ; th century, continuetodosotoday. and rns and processes; (3) theuntanglin (3) rns andprocesses; ecular and microscopical and methods ecular microscopical se to sample the ocean floor (B floor ocean tosamplethe se la Infusoria (Müller 1786). In the1800’s, 1786). Infusoria (Müller la to great improvements that were expanded upon by fora uponby expanded thatwere s. think thattheAlthough we datafromthe fieldisrelatively osphere. Majorquestions about al questions in biology, (2) ev al questionsinbiology, (2) nard (1855–1954) describeda nard (1855–1954) eafloor sedimentsamplesfromthe hers made numerous remarkable made hers microbial eukaryotic diversity. microbial time Christian Gottfried Ehrenberg (1795-1876), time ChristianGottfriedEhrenberg (1795-1876), (1632-1723), Louis (1632-1723), (1645-1723), and Joblot Otto by Dujardinin Félix 1835, and Alcide d'Orbigny itats. In 1900’s, the itats. Skuja(1892-1972), Heinrihs unanswered, largely because data obtained from from obtained data largely because unanswered, el (1834-1919), Georg Klebs (1857-1918) and (1834-1919),Georg (1857-1918) el Klebs th pand the tree of eukaryotes; (2) the recognition recognition (2)the eukaryotes; of pand thetree e studies performed in in performed e studies century, the remain least protists explored about protistan diversit processes; and (4) the evaluation of protist evaluationof (4) the and processes; 976). Joblot’s reflected of protozoa drawings tiful drawings many of free-living protists. (Fig. 1),andMüllerproducedthefirstbook “field protistology” as the exploration, ; microscopy; molecular phylogenetics; phylogenetics; ; microscopy; molecular , many majorcontributions ofwhommade ht howfieldworkinprotistology provides which allowed the first discoveries of of firstdiscoveries the which allowed r exploratory research inprotistology. research r exploratory The

, or fungi rady 1876).The rady naturalists Joseph g of ecological interactions and g ecological interactions of aluate why data from the field thefield from data aluate why offer the most promising path offer mostpromisingpath the nd illustrated dozens ofnewnd illustrateddozens suchasHenry minferologists the diversity,ecology, and observations ofprotists, observations HMS Challenger y is notably illustrated the field so far have the fieldsofarhave cruise cruise also under-sampled, although protis 2011; Hege documented (Fontaneto et al.2012; Hanson unknown. Also,theprocesses thatgovern thei al. 2011), et 2006;Heger al. 2010;Foissner ge the pa biogeographical for evidence compelling is there distributions (Foissner 1997;Foissner 1999; Fo 1999). assumesthatatleas view The second formsallo produce resistant to 1mm), andcapacities th that allmicroorganismsare ubiquitousbecause contentious issue be a living protistshasbeen environments outsideEuropea se biodiversity been conducted in have surveys addr questions inprotistology fully be cannot This article is protected bycopyright. reserved.All rights cycles. biogeochemical geneother studiesmight our seriously bias 2012) (http://www.lternet.edu/). prot Neglecting questions abouttheimpacts ofla Long Term (LTER) Research Ecological Network groups oforganisms. compared toother inst For scales.In aquatic terrestrialand ecology, also protistshave received relatively attention little and, thus,helptoch library barcode reference http://www.barcodeoflife.org) f willundoubtedly Group (Pawlowskietal.2012)oftheCons etal.2013),the Database (Guillou AlgaeBase (G groupsother oforganisms. Fortunately, ambitious Bacillarophyta instance, species. For protist estimated numberof actua the between importantdiscrepancy by the Accepted Article speciesandtheto protist number ofdescribed actual the between gap a significant is there that protistologists among consensus a strong (Boenigk etal.2012).Eventhough itis important to limited by available data and the way data these are interpreted by different protistologists groups indifferent of ofspecies the totalnumber for estimates data. Therefore, phylogenetic molecular with recognized ofspecies overestimation basisofmorphological the on solely recognized protist species data onthe isdependent kindof tocharacterize used them; forinstance, species de been have 6,000 species 10 millionspecies,only parasite foundin gapcertainly is a total outof 3,500 ciliatetaxa (Mann and species) (i.e., only 10,000to20,000 Protists are also poorly studied across space acrossspace Protists arealsopoorly studied In molecular barcode reference libraries,pro reference In barcode molecular

comprises about 200,000 taxa but only tent a but about200,000taxa comprises

nd North nd North (Adl et al.2007) America of 30,000 estimated species have been described but the biggest biggest butthe described been have species estimated of 30,000 s like apicomplexans where outof where s likeapicomplexans ke acidification,clim tan species often exhibit very exhibit pa often tan species tal number of protist species on Earth. on Earth. ofprotistspecies tal number tween two opposing views. The first view assumes assumes twoopposingviews. tween The firstview ral understanding of f understanding of ral Droop 1996, Norton et al. 1996). Similarly, 1996,Nortonetal. Droop only aracterize protistan diversityat finetaxonomic aracterize issner 2008; Vyverman al.2007). et Although l number of described species and the overall overall and the species described numberof l essed. protist few noor global scale, At a protists are not necessarily comparable and are are and comparable protists arenotnecessarily istan communities in these experiments and communitiesintheseexperiments istan r diversity and biogeography remain poorly r diversityremain poorly and biogeography t some of them have limited geographical limitedgeographical t someofthemhave ance, they are generally not integrated in the the in integrated not generally are ance, they RibosomalReference projectsliketheProtist traits may be either an either traits may be eir huge populations, small cells sizes (i.e., < (i.e., populations, smallcellssizes eir huge ortium for the Barcode of Life of (CBOL, ortium fortheBarcode uiryor theProtist andGuiry 2013) Working experiments, which aim whichaim experiments, ographical distribution of most taxa remains remains distributionofmost taxa ographical veral regions ontheplanet,especially in veral regions it is thought that the photosynthetic protists it isthought protists thatthephotosynthetic and time, andtherefore, and time, scribed (Adl et al. 2007). The recognition of of The recognition 2007). et al. (Adl scribed w for a globally dispersal (Finlay and Clarke and Clarke (Finlayglobally dispersal w fora acilitate thedevelopm treatspeciesestimates tterns in different groups of protists (Boo et groups ofprotists(Boo different tterns in tists are also underrepresented compared to comparedto tists arealsounderrepresented ate change or diversity loss (Knapp et al. ate change ordiversity loss(Knappetal. r et al. 2013). et r protistsare scales, At local

h of its diversity has been described described been h ofitsdiversity has . distributionoffree Thus, the an estimated number of 1.2 - - estimated numberof1.2 an tchy distributions, especially distributions, tchy ood web processes and webprocesses ood underestimation or an with caution,thereis several fundamental several to address important to address ent ofaprotistan 2013). Guillouet etal.2010; environments (Glücksman heterotrophic protistsis alsoakeyrequireme YubukiLeander 2013,Zhao and etal.2012). bi The stateswithin the euka ancestral overall treeof apusomonads, (e.g., so- of ultrastructural features 2008; Leander and Yubuki Reconstr etal.2009). Glücksman etal.2011; Kolisko etal.2010; Walk ofmajorlinthe phylogenetic relationships ultrastructural evolution, c have fornicate excavates al. 2010). Culture-independent assessments of di assessmentsof al. 2010).Culture-independent morphologicalet al.2005;Boen (Boenigk traits the inherentdifficultiesof cultiv protiststhatmightliving play heterotrophic a cruc This article is protected bycopyright. reserved.All rights identification andcultivation of cellular, ecological, behavioraland spite levels.In ofthe difficulties associatedwith the th taxa uponreference dependent almost entirely et al.2001;StoeckGarcía etal.2009).However, pr heterotrophic uncultured fluorescent wh sediments in soilsandmarine , cultivation anddescriptionof evolutionary biology, ecology, andmo andcell a diversity the point forunderstanding cu ofstable establishment environments andthe 1994; Rimaand Aaron 2007; Weisse Accepted ArticleOrsi etal.2011; Takishita etal. distributions(Foipatchy have and low abundance yetMost protistshave bedi to Free-living heterotrophic protists protists, their spatial distributionsand their temporal dynamics. (http://www.biomarks.eu) toincreas needed are (http://oceans.taraexpeditions.org) (Kar organized onerecently by the campaigns suchas TARALarge-scale sampling relatively documented, poorly particularly insoilecosystems (Fig. (Mitchell et al. 2011). 2E) 2010; Fenchel andFinlay1986; Nolteetal.2010). Ho greatlyam candiffer and pronounced can alsobe

New cultureNew facilitate indiverse strains furtherinvestigations such disciplines as

Collodictyon, Mantamonas, Palpitomonas, , Mantamonas,Palpitomonas, , Tsukubamonas in situ hybridization (FISH) have contributed (FISH) hybridization contributed have Breviata -prokaryote sy eukaryote-prokaryote otistan biodiversity inaquatica called “sister-less”or scovered, especially free-living he free-living especially scovered, ontributed significantly to an improved understandingontributed significantly toanimproved of and 2007). The discovery, characterization 2007). The novel or poorly characterized pr novel orpoorlycharacterized ating them, their smallcellsizes, ating them,their ere spatial heterogeneity is co is heterogeneity spatial ere

heterotrophic ,sing heterotrophic Collodictyon 2008). In to spatialvari addition nd biology of heterotrophic protists. of heterotrophicprotists. nd biology senti etal.2011)senti BIOMARKS or ryotes (Kimetal.2006,Rogerryotes andSimpson2009, eages of eukaryotes (Brugerolle etal.2002;eages (Brugerolle ofeukaryotes ltures remains a fundament a ltures remains igk 2008; Chantangsi and Leander 2010; Jost et Leander 2010;Jost igk and 2008;Chantangsi e our knowledge about the overall diversity of diversityabout theoverall of ourknowledge e versity based on environmental PCR surveys or PCRsurveys or onenvironmental based versity ong ecosystems and taxon groups (Behnke et al. groups(Behnke taxon and ecosystems ong ) has facilitated inferences about the deepest deepest aboutthe inferences facilitated has ) ucting the molecular phylogenetic position and positionand phylogenetic molecular ucting the er etal.2006; Yabuki etal.2010;2011; Yubuki ssner 1999; Lèfevre ssner et al.2008;Lin et al. 2012; mbiotic relationships, or lecular biology. thediscovery, For instance, ial role in ecosystems are also complicated by alsocomplicatedby are ial roleinecosystems nt for understanding their role in various their roleinvarious nt forunderstanding al. 1999; Matz et al. 20 et al. 1999;Matz analyzing these data fromfieldsurveys is thesedata analyzing odiversity and taxonomic at have already been characterized at the at characterized been already at have “orphan” lineages of heterotrophic protists heterotrophic protists lineages of “orphan” wever, varia temporal

greatly to our overall understanding of understanding greatly toouroverall nd terrestrial environments(López- nd terrestrial mmon (Engel et al. 2012; Fenchel Fenchel al. 2012; et mmon (Engel terotrophic species that occur in occur speciesthat terotrophic otists, suchasapusomonads, le-cell isolatesfromnatural le-cell and the lack of conspicuous of lack the and ability, variations temporal , and identification offree- ganelle evolution,and ally important starting 02; Seenivasan et al. 02; Seenivasan tions ofprotistsare characterization of characterization ,

and 1993). For these reasons, theuseof 1993). Forthesereasons, al. et Broers (e.g. associations facultative of case the in partners more or ofone as elimination relationshipscould symbiotic the Furthermore, (Kimetal. 2010), ha of endosymbiotic and thefreshwater aureus Somelow-ox et al.2000). environments (Bernhard addition, protist-bacterial a varietyasso of an providing hostby acantharian benefits the th where waters, oceanic oligotrophic surface between theradiolarian andthehaptophyte Acantharia carbonto th fixed , whichprovide photosynthetic protists etal.1999;Foster (Carpenter contribute significantly endosymbionts, which such as in For instance, 2010). Melkonian and (Nowack particular, appear many harbor microbial to consortia based onthelimiting nutrient factors et al.2009). for surveyed theecosystems Among 2011a;al. 2009;Johnson 2012; Gastet 2011b; 2010;Stoecker Johnson Melkonian and Nowack ha on protistsymbioses reviews comprehensive al. et those organisms (Graham involved This article is protected bycopyright. reserved.All rights Accepted tools, newpreservation Thompson etal.2012), (e.g. cytometry cell single such as on-board advancements, technological locations, remote in those especially symbioses, ofprotist andcharacterization identification the to impediment otherorganisms. with that formsymbioses Wh protist cond culturing standard under symbiotic microbes partiallyexplained by difficulties the and challenges associated withisolating culturing and and Melkonian2010),themajority themst of unclear. are associations these the al. 2003),although et (Baker mine drainage and acid Inouye 2006), and (Okamoto beaches al. 1991),sandy (Stoecker et 2007), estuaries in other ecosystems, protists occurinmany and/or epibiontsontheirsurface possess many Finlay syntrophy In and (Fenchel 1995). termite contactwiththehy inclose methanogens occur as such al.2011a).Inanaerobic 2010; Edgcombet produ sulfide compounds,and/orremovehydrogen sulfid reduce to appear epibionts,which bacterial Articleassociations haveimporta Many formsymbioses withother protists protists,bacteria, animals, orfungi, and , such Protists living with other organisms While a growing number of protist symbioses are being discovered each year (Nowack year (Nowack each being discovered are symbioses ofprotist agrowing number While Hemiaulus , Bihospites and harboring the green harboring alga the green such astheciliates nt roles in ecosystem functioningnt rolesinecosystem a

Petalomonas sphagnophila et al. 2007). In etal.2007). oligotrophic bacati Rhizosolenia fresh fieldsamplesisoftennece , and mariagerensis associate with nitrogen-fixing cyanobacterial associate cyanobacterial withnitrogen-fixing cluding eutrophic oceanic surface waters (Gomez waters surface oceanic eutrophic cluding ile such sampling requirement has been a major a hasbeen requirement ile such sampling ciations have been observed in low-oxygen in low-oxygen observed havebeen ciations 2002; Paracer and Paracer 2002; Ah be altered during the culturing procedures, such such culturing procedures, the during altered be drogenosomes of the for hydrogen-based ciliate forhydrogen-based ofthe drogenosomes tioxidant protection (Decelle et al. 2012). In al. 2012). et protection(Decelle tioxidant e heterotrophichost(Glime2012). Associations e pigmented algal presumably presumably endosymbiont algal e pigmented ve not been cultured despite numerous efforts.ve notbeencultureddespite ecophysiological factors that facilitate much of facilitatemuch of factors that ecophysiological ill remainunknowntoscience. This may be guts, anaerobic and parabasalids and oxymonads anaerobic guts, Colpoda to the nitrogen budget of the diatom host ofthe budget to thenitrogen ve been published elsewhere (e.g. (e.g. elsewhere published ve been Anderson Nephroselmis e toxicity in the host by consuming sulfur or sulfuror consuming by inthehost e toxicity intracellular bacteria. Symbioses involving bacteria. intracellular itions. For example, the marine marinekatablepharid the itions. For example, and single-cell genomics are opening up up opening genomics are andsingle-cell nutrient-poor surface ocean waters, oceanwaters, nutrient-poor diatoms surface ygen dwelling protists like the euglenozoans euglenozoans ygen the protistslike dwelling ced by host , (Breglia et al. (Breglia etal. ced by hosthydrogenosomes, protist diversity, in waters, oligotrophic associating withseveral different kinds

Phaeocystis and Sphagnum nd evolutionary di nd evolutionary Plagiopyla frontata sp. (Okamoto and Inouye 2006) Inouye and 2006) sp.(Okamoto Prorodon ssaryfor thestudy ofprotists are thought tooriginate arethought in are covered with numerous withnumerous are covered -dominated peatbogs, non- madjian 2000). More madjian 2000).More carry green algal carrygreen versification of versification , intracellular This article is protected bycopyright. reserved.All rights animalsha important economically Until relativelyrecently, few other parasitic than protists thoseaffecting and/or humans Parasitic protists ecosystems. toinvestigate wider a opportunities unprecedented important forourunderstanding of multiple and/or large-scale,efficient and rapid screeningDNA of which isparticularly samples, labor between shared and isolated, stored easily protistsusing ofparasitic of molecularbiology American Type (ATCC).Collection Culture aspects various study can However, one present, at usually hadtobeisolatedandintroducedinto pathogenicity, ultrastructure,aswell lifecycle, 12 (MAST 12) as a close relative of the human parasite, parasite, human the of relative close as a 12 (MAST 12) clades oftheobligatoryparasitic groups,promin se ofenvironmental analyses al. 2008).Recent particularly successful in thea in successful particularly and Quake 2011),promise toprovide critical hold insight forparasitism, into the adaptations eraofwhole-genome the in accessible protists, geneticdiscriminatorywidemap their unexpectedly diversity.power to genomes parasiticThe of possess awealthofdistinguishing morphological et al. (Pawlowski diversity eukaryotic all extant Moore etal.2008)and,evenmoreimportantly, i organisms and asrelativesofapicomplexans and the chromerids revealed also analyses molecular dependent Culture Accepted etal.2013). (Flegontov trypanosomatids novel monospecific clade identifieda recently the same techniques time,revealed molecular greater anticipated than far genetic diversity of “one host- based onthe predicted than whatwas from different continents, strongly indicating the global that diversity ofthese protistsis lower encount was species same the instances 2013). insome cladesast While trypanosomatid major insight intothe varied biodiversity landscape ofthese important parasites (Maslov et al. relatively limited inscope, a re exciting theory be seemed hardly to testable inthe foreseeable However, future. despite being isolatedfrominsectswasavaila handful ofspecies number ofspeciestheseprotistsmustr combination withpostulated high hostspecificity ofinsect-dwelling trypanosomatids means the features. Ayears ago, few it was proposed that Trypanosomes, andre leishmanias well-studied groups ofhistorically on two Article

The power of molecular techniques combined with field collections can be demonstrated demonstrated be can collections field with combined techniques molecular of The power spp. in the thoroughly and long studi and long thethoroughly spp.in bove-mentioned widespread protists. newed emphasis on fieldcollecti emphasis newed lated flagellates areknownforth lated flagellates the diversity and evolutionof the diversity ve been examined indetail. examined ve been each millions(Stevens 2001). At thattimeonly a just tiny amounts ofDNAjust RNAand/or be thatcan , respectively (Koliskoetal.2010;diplomonads, respectively atories. Moreover, molecular methods now allow atories. Moreover, now methods molecular one parasite” paradigm (Votýpka paradigm parasite” one et al.2012). At ent examples being mari ent examples 2012). In general, most pa culture or obtained from a collection, suchas acollection, from orobtained culture quence data identified novel free-living sister free-living sister identifiednovel quence data as itsmolecularandcellbiology, theparasite features, yet provide the features, moleculartechniques sequencing and single-cell genomics (Kalisky genomics (Kalisky andsingle-cell sequencing ndicate thatprotistsmay constituteupto¾ of ble in culture (Maslov et al. 1994) and such an al. 1994) andsuchan ble inculture(Maslovet parasites – trypanosomes and plasmodia. andplasmodia. –trypanosomes parasites rict hostspecificity holds,inseveral indeed ered in different heteropteran hosts coming coming hosts heteropteran different ered in the species abundance of insect hostsin insect of abundance thespecies ed tse-tse flies (Adams et al. 2010) and and al. 2010) et flies (Adams ed tse-tse that represents a sistergroup a toallthat represents range of protist associations in diverse associationsindiverse ofprotist range

Blastocystis ons (Fig. 2F) already provided provided already 2F) ons (Fig. (parasitic) protists(Koliskoet Typically, for thestudiesof eir dearth of morphological eir dearthofmorphological ne stramenopile cluster ne stramenopilecluster (Massana et al. 2006) et al. (Massana rasitic protists do not not rasitic protistsdo -like .

This article is protected bycopyright. reserved.All rights interest intheseorganisms contributedirectly a sampling, the comparatively small active number “field of protistologists”, and limited public species, difficulties and associatedwith cultivation. Furthermore, challenges associated with small bodyidentify oftheir because their size, inmolecularapproaches developments and recent ofelectronmicros centuries ago, theapplication data remains limited. First ofall, despite the invention oflight microscopy more than three methodological ch several andconceptual There are Why are data from protistan field studies limited? reach. recently,wereallow parasitic until studies protiststhat of another, or reason for one beyond other primates(Prugnolle million peopleannually, may have proposal thatthehumanpernicious the by to apesled inhabited big environment the in collected analyses offeces Extensive animals, for and infactotherendangered toinve an avenue samples opened ways. All sampling andisolation of amyriad in accomplished canbe sediments shallow or nearshore and soils 2A), (Fig. waters ofisolating nucle cultures, orforthepurpose for sampling field to approaches many are There sea deep the in casestudy challenges: Sampling protistological disciplines. Accepted typically arethen Samples 2C-D). (Figs. samplers rosette Niskin via surface the brought to water whereby (Fig. 2B) operations hydrocasting Current studies ofpelagic microbial communities marine rely heavily onship-based 2011c). al. et andintegrity (Edgcomb state metabolic cell impact canpotentially pressure that pro because great, particularly concernsare these et al.2011 2001;Orsi et al. Lopez-Garcia 2008; Edgcomb (e.g., without closeculturedrelatives co of diverseprotist signatures andimages ma mesopelagic andbathypelagic material priortoinitiating experiments. protis the of recovery in biases to approaches, (particular many protists growth of the encourage ofdifferentprotist ta preservation di fromthe These biasesrange unknown extent. Article The finding that The finding that Deep ocean realms remain largely remain understudied realms Deep ocean et al. 2011). al. et pow the Incase, any

methods havethe potentialtoin xa formicroscopy,xa replicating or stigate its diversity in big apes and monkeys (Liu et al. 2010), al. 2010), (Liu et andmonkeys apes inbig stigate itsdiversity been originally (Rayner fromgorillas etal.2011)acquired or rine water columns andsedime columns rine water Plasmodium falciparum DNA can be PCR-amplified from field-collected fecal fecal DNA field-collected from can bePCR-amplified mmunities that include many taxonomic groups mmunities thatincludemany taxonomic ic acids orotherbiom ic ts or their biomolecules from theor their original biomolecules samplefrom ts which tissue samples are virtually inaccessible. inaccessible. arevirtually samples tissue which patchy distributions,thelow abundanceofmany copy to biological materials inthemid1900’s,copy tobiological materials ). When it comes to sampling the deep ocean, ocean, deep itcomestosampling the When ). et al.2011a; 2011b;Lopez-Garcia and Epstein tists are also exposed to significant changes in changes exposed to arealso significant tists ly in isolation) with currently known culturing culturing known in isolation)withcurrently ly nd indirectly tothe lack samples from various depths in the ocean are are intheocean depths samples fromvarious allenges that help explain why thathelpexplain allenges field protistan , protistsremain difficult todiscover and isolation of protists in enrichment or pure pure isolation ofprotistsinenrichmentor fficulties associated with adequate cell cell fficulties withadequate associated regarding protistdiversity. regarding Studies of

, responsible for the of over a a thedeathofover , responsiblefor er ofPCRandsi troduce biases tosomeusually in situ nts to datehaverevealed olecules. Sampling surface Samplingolecules. surface conditions enough to conditions enough of data in several ofdatainseveral ngle-cell genomics particularly oxidation-reduction oxidation-reduction particularly depths. greater when samplingpotentially compounded duringfiltration. This problem isparticularly se ship's laboratory. Variable of unknownfractions and environmental conditions. Theref of studies tointegrated studies ofdiversity environments. structure a community undetected) (often in states. canmanifest This ofphysiological modification dramatic in deck canresult ship's the to environment the from samples the ofbringing act very the where studies expression be betterpositionedtoexamine situ deca a over datain and microscopic molecular firstnovelclassof imagesmicroscopy ofthe ce protist greater magnitude in situ oflivingshowed fewsigns eukaryotes. samp When Basin, Venezuela, 2011), al. et (Orsi community a diverse indicated signatures molecular where deeperwithinthe andfromtwodepths interface below~250m. Microsc interfaces samplinga only fraction ofthe recovers total due tounnaturalandchanging longer preservation half-lives, delayed ofsuch samples can stillbe an issue ifcell integrity islost significantly their to andrRNA due suchbiases to identifiers) susceptible less are as phylogenetic samples came. DNAWhile (notan indicator ofcell viability butselected genes commonly used plastic Niskinbottle walls]) are often significantly altered from the environment fromwhich the This article is protected bycopyright. reserved.All rights Accepted by O contamination [sample state redox light, temperature, Articlewater reach samples are deck oftheshipand the processed, sample conditions (pressure, sample ina Niskinbottle at inthe depth ship's and filtration/fixation laboratory.By the time the of whenupwards studies ofgeneexpression, 2006; Selinger etal.2003; Wang et al.2002)it hal short typically the Given balance. climate are essentialtoourunderstanding ofecological columnox ofwater levels responses tochanging of marine microbial ecology, butitismore lim sampling willalways Niskin molecular analyses. culture-based,functional phylogenetic, and/or bottle drawn forsubsequent the from Niskin water column samplingmicrobiological and preservationfor studies are developed, we will using a pre-programmed remote using pre-programmed a Many microbial eukaryotes are extremely sensitive tophysico-chemical changes, Research trends in marine microbial ecology are transitioning from purely descriptive descriptive purely from transitioning are ecology microbial marine in trends Research ll count(unpublished protist populations and their activities in the deep sea. deepsea. protist populationsandtheiractivitiesinthe conditions during transportfrom (redox) state, pressure, and temp state,pressure, (redox) ope observations of fixed Niskin samples from the oxic/anoxic Niskin samples offixed fromtheoxic/anoxic ope observations ore, systems-level investiga ore, systems-level lterations, foranox lterations, particularly water sampler, water minimum twoordersof a they revealed in situ 30-45 minutes can pass between enclosure of the of enclosure minutescanpassbetween 30-45 in situ f-life of mRNA of f-life mi (e.g. de (Orsi et al. 2012). et (Orsi de for new technologies As ciliates tobedocumente ciliates phenomena intrinsic to ocean productivity and phenomena intrinsictooceanproductivity anoxic (340mand900zone m)intheCariaco anoxic ygen deficiency,ygen including studiesofprotozoa, iting rate process for me vere for the morefragilefor the vere marine protistsand have a role in oceanography and some aspects andsomeaspects have aroleinoceanography activity and microbial responses to changing changing responses to and microbial activity is especially difficult to conduct meaningful difficultis especially conductmeaningful to les from deeper anoxic waters were preserved preserved were waters deeperanoxic from les genetic material from lysed cells can be lost be cells can lysed from genetic material community from anoxic watersandredox community fromanoxic data) and provided scanning electron electron scanning data) andprovided

2 dissolved in & diffused dissolvedin the from tions of microbial community microbial community tions of the ocean environmenttothethe ocean ic and/or deep-water column column ic and/ordeep-water erature; consequently Niskin consequently erature; nutes) (Andersson et al. et al. nutes) (Andersson d onthebasisofboth d asurementsgene and in This article is protected bycopyright. reserved.All rights Firstly, beovercome. need to Next-Generati biomonitoring approaches. However,address inorder these to several objectives, obstacles still evaluation ofprotists’ ability adaptchangingclimates,(5) to to and thei functions, and and interactions of ecological ofnovelevolutiona therecognition eukaryotes; (2) of tree the expand that lineages ofnew discovery (1)the to contributing are These newdata insi opportunity toprovideenormous unprecedented withhigh-end combined molecular approaches organisms fieldsurv studied poorly from new and It isa very exciting time forresearchers interested inprotists. Constant efforts forcultivating Future perspectives andconclusions LITERATURE CITED comments onthismanuscript. providing numerous PA00P3 for editor associate one and referees anonymous thankthree we 145374).Finally, Institute for issuppor Research. T.H Advanced British Columbiaforfunding thissymposium. B. as the Tula Foundation’s Centrefor MicrobialDi projectsinprotistolo support ambitiousresearch ofbiodiversity.understanding Finally, thisadvo essentialtodifferent is exploratory research research forhypothesis-driven showing preference community.collection by thebroader Fourthly, at deposited inpub should bemoresystematically lightands sequences, extraction, datasets. Thirdly, and material databases willimprove studies, andother informative culture-based differentgeographical ecosystems regions, and time-scales. These efforts willnotonly lead to it iscrucial toincrease protiststhrough sampling large-scale the of campaigns samplingacross ofsamples toimprovetheanalysisexaminations Furthermor assignments. taxonomic must beimprovedtoproduce l Adams, E. R., Hamilton, P.Adams, E.R.,Hamilton, & Gibson, B. W. C.2010. celebrating trypanosomes: African Accepted Norway.Protist 2012meeting, Oslo, WeIn the thank thediversity,understanding and evolut ecology This review isthe direct result ofthe “Field symposium protistology: cornerstone for ACKNOWLEDGMENTS instance, willintegrateprotis projects which,for Article diversity. Trends Parasitol., taxonomic assignments taxonomic onger read sequences of higher ofhigher sequences onger read data obtained directly or indire or directly data obtained canning microscopy pictures, fresh and cryopreserved cultures) cultures) microscopy freshandcryopreserved canning pictures, 26:324-328. e, these approaches need to be paired with microscopical withmicroscopical paired tobe need approaches e, these

on sequencing techniques, techniques, on sequencing but resulting genetic da resulting but ts in long-term and multi-trophic experiments. and multi-trophicexperiments. ts inlong-term disciplines inprotistology, sound any andto andinterpretationofmo cacy will help to ensure that funding agencies thatfundingagencies will helptoensure cacy gy and facilitate collabo gy and ion of microbial eukaryotes” held during the heldduring ion ofmicrobialeukaryotes” microscopical methods,together an represent ted by the Swiss Science Fundation (project Fundation ted by theSwissScience lic collections to improve access to a wider a wider to access lic collectionstoimprove r roles in larger ecosystem processes; (4) the the (4) processes; larger ecosystem r rolesin , itis important toemphasize andpromotethat of sequences recovered in Next-Generation inNext-Generation recovered of sequences a time when funding ag atimewhenfunding eys and the use of efficient and inexpensive efficient and of theuse and eys ry patterns and processes; (3) the untangling (3)theuntangling and processes; ry patterns S.L. and J.L. are fellows of the Canadian oftheCanadian fellows are and J.L. S.L. versity andEvolutionattheUniversity of ternational Society of Pr of Society ternational ghts intofundamentalquestionsinbiology.

quality that willallowforbetter quality that ctly from the field (e.g., DNA thefield(e.g., from ctly

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FIGURE LEGENDS Yubuki,Leander, N.& of B. andmolecularphylogeny S.2008.Ultrastructure Yubuki,Leander, N.& treeof B.S.2013.Evolutionofmicrotubule organizing acrossthe centers Yabuki, A., Nakayama, T., Yubuki, N.,Hashimoto, T., Ishida,Inagaki,I. & K. Y. 2011. Zhao, S., Burki, F., S.,Burki,Zhao, Keeling, P. J., Brate, & Shalchian-Tabrizi, Klaveness,D. J., K.2012. Zaikova, E., Walsh, D. A., Stilwell, C.P., Mohn Yubuki, N.,Edgcomb, V. P.,Le M.& J. Bernhard, Yabuki, A., Inagaki, Y. & Ishida, K. 2010. Whitney,F. Persistently 2007. Freeland,declining &Robert, M. A., H.J. oxygen levels inthe Weisse, T. 2008.Distribution anddiversity a of for Marine-Earth Science and Science for Marine-Earth Technology, JAMSTEC) Bamfield, Pacificcoast west ofCanada. Fig. protists. Fig. F in the Swiss Alps toassess the patterns oftestateamoeba diversity along analtitudinal gradient. north western PacificOceancarousela Niskinbottles via system. Wang, Y.L.,L.,Liu, C. Storey, D., J. Tibshirani,& Herschlag, R. Brown,P. D. J., O.2002. This article is protected bycopyright. reserved.All rights Walsh, D. A., Zaikova, Howes,C.G., E., Song, Y. C., Wright, J., J. Tringe, S.G., Tortell, P. D.&

AcceptedInsects are order. collected toassess parasitic protist diversity in their forest). (Ecuadorian Article 2. 1. “Small animals” drawn by Louis (J drawnby Jablot “Smallanimals” Photosillustrating field protistology research. eukaryotes. for the early evolution in"Discoba". Tsukubamonas globosa 538. Collodictyon Protistol., minuta relate possibly heterotroph branching 1568. Columbia. Microbial community dynamics ina seasonallyInlet, fjord:Saanichanoxic British euglenozoans withepibiotic bacteria. of phylogeny interior waters the eastern of subarctic Pacific. 99:5860-5865. perspective. perspective. inmRNA specificity functional Precision and decay. oceanic dead zones. zones. dead oceanic 2009.Metagenomeofavers Hallam, S.J. : An enigmaticmicroeukaryote frommarine interstitial environments. 44:241-253. Environ. Microbiol., The Plant J., Plant The Biodiver. Conserv., -An ancient lineage in the tree of eukaryotes. eukaryotes. of tree the in lineage -An ancient

Calkinsia aureus Science, n. gen., n. sp., a novel excavate flagellate possibly holding a key holding akey flagellate possibly excavate a novel n.gen.,sp., 75:230-244. 326:578-582. 326:578-582. 12:172-191. 17:243-259. B. : cellular identity of a novel clade of deep-sea ofdeep-sea novelclade ofa identity : cellular Oceanographic research vessel, Mirai (Japan (Japan Mirai vessel, Agency research Oceanographic J. Eukaryot. Microbiol.,J. Eukaryot. BMC ,BMC d to d to orHacrobia. quatic protists:anevol ander, S.2009.Ultras B. , W. W., Tortell, P. 2010. D.& Hallam, S.J. ablot 1718); one of the first illustrations of ablot 1718);oneofthefirstillustrations atile chemolithoautotroph from expanding chemolithoautotrophfrom atile Prog. Oceanogr., C-D.

A. Water the in arecollected samples Collection ofwater samples in gen. nov.: etsp. A noveldeep- 9. Proc. Sci.U.S.A., Natl. Acad. E. 58:319-331. Mol. Biol. Evol., Mol. Biol. Soil samples are collected collected are samples Soil 75:179-199. 75:179-199. utionary and ecological utionaryecological and tructure and molecular andmolecular tructure Protist, Stephanopogon 29:1557- 161:523- Eur. J.

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