Protist, Vol. 164, 842–860, xx 2013

http://www.elsevier.de/protis

Published online date xxx

PROTIST TOOLS

A Short Guide to Common Heterotrophic

Flagellates of Freshwater Habitats Based

on the Morphology of Living Organisms

1

Alexandra Jeuck, and Hartmut Arndt

Department of General Ecology, Zoological Institute, Cologne Biocenter, University of

Cologne, Zülpicher Straße 47b, D-50674 Cologne, Germany

Introduction Kim and Archibald 2013). In contrast, the domi-

nant taxonomic groups within different marine and

Heterotrophic flagellates (HF) are very likely the freshwater pelagic communities (e.g. stramenopile

most abundant eukaryotes on Earth, hundreds of taxa, dinoflagellates, choanoflagellates, kathable-

specimens occur in each droplet of water even pharids) and benthic communities (e.g. ,

in groundwater and the deep sea. As the main free-living kinetoplastids, cercozoans) seem to be

feeders on bacteria they play an essential role in surprisingly similar (Arndt et al. 2000). Their modes

aquatic and terrestrial food webs (Arndt et al. 2000; of movement as important taxonomic character-

Azam et al. 1983; Bonkowski 2004). In addition, istics comprise gliding or free-swimming forms or

they can act as important herbivores (Arndt and they may be temporarily or permanently attached to

Mathes 1991; Nauwerck 1963; Sherr and Sherr a substrate (Fenchel 1987). Feeding modes include

1994), detritivores (Scherwass et al. 2005) and true filter-feeding (e.g. choanoflagellates), direct

osmotrophs (Christoffersen et al. 1997; Sanders interception feeding (e.g. chrysomonads) or rapto-

et al. 1989; Sherr 1988) as well as (Bird rial feeding (e.g. most benthic forms) (Boenigk and

and Kalff 1986; Sanders 1991). The relative contrib- Arndt 2002; Fenchel 1991).

utions to these different modes of feeding can vary One important prerequisite to estimate the role of

within taxonomic groups and even within one and HF for the flux of matter in ecosystems is the deter-

the same organism, e.g. Ochromonas sp. (Jones mination of their abundance. In the early days of HF

2000; Wilken et al. 2013). Furthermore, bacterial quantification, the so called “HNF” were counted

communities are not only grazed by protozoans exclusively in fluorescently stained fixated sam-

but are also structured by protistan grazers (e.g. ples (e.g. Porter and Feig 1980). However, fixation

Boehme et al. 2009; Güde 1979; Jürgens and Matz bears the problem that many species are disrupted

2002; Pernthaler 2005). upon the fixation process (Choi and Stoecker 1989;

HF are a very heterogenous group with an Sonntag et al. 2000) or are misidentified as small

␮

enormous size range between 1-450 m (some naked amoebae, zoospores, yeasts, or disrupted

␮

authors refer to the species smaller than 15 m as cells from a range of eukaryotes, since flagella are

heterotrophic nanoflagellates “HNF”, Arndt et al. not always adequately preserved (Patterson and

2000). High tolerances to changes in salinity Larsen 1991). Especially benthic HF are affected

allow several species to live both in marine as by the above explained problems because they are

well as in freshwater habitats, though several additionally masked by sediment particles. Further,

phylogenetic studies have also indicated clearly large HF can easily be overlooked on membrane

separated marine and freshwater clades (e.g. filters due to their low contribution to total HF abun-

dance and their special sensitivity to fixatives. This

1 is crucial since they may contribute to about half of

Corresponding author; fax +49 221 470 5932

e-mail [email protected] (H. Arndt). the HF biovolume (Arndt et al. 2000). Therefore, the

© 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.protis.2013.08.003

Guide to Free-living Heterotrophic Freshwater 843

JJaJacobcobidsbidds Free-li v ing

kinetopla sds

Euglenids

BicoBi soecidsid Diplomonadsp

Chrysomonads PPreaxostyla l

Discoba

Mala wimonas Ciliophp ryidy s Metamonads

Dictyostelians

Algae – group

(Rhodoph yceae, Chloroplasda, Mulcilia

GlGlaukophph ytay ) ?

Tubulineansb l

Dinofla gellates

Stramenopiles ? Phalansteriids

Alveolates FunF gii

CCercozoans Nucleariids

Ichthyospo reans

Foraminiferan s

Polycysneans Metazoans Choano flageg llates CCe rcomonadsd

Spongomonads ?

PROTI STS OF UNCERTAINAIN POSITION

Thaumatomonad s

Cryp tophyceans Apusomonads Spironemids

Glissomonads AAncy romonadsd

Kathablepharids Phyy llomonas

Cryomonadsd Collodictyonids

Figure 1. Selective overview of the systematic position (according to Adl et al. (2012) of flagellate groups

considered in the guide (see Charts 1–15).

taxonomic resolution of fixation processes is often freshwater flagellates due to its polytomous organi-

very low, most quantitative studies even ignored the zation, the help of drawings and video sequences

variety of HF. (Supplementary Material). According to our experi-

In contrast, live counting is an essential and reli- ence in quantitative studies of morphotypes, about

able method for determining HF abundances. It 80-90 per cent of the dominant heterotrophic flag-

allows a combination of taxonomic and quantita- ellates found in freshwater habitats should be

tive studies for understanding the role of HF in identifiable at least to rough taxonomic categories

microbial food webs (Leadbeater and Green 2000; (and functional guilds). The term morphotype is

Patterson and Larsen 1991). Unfortunately, the tax- used here for taxa that can be differentiated by

onomic resolution of HF studies is hampered by the means of their morphology when only analysed

fact that the literature is often focused on only a few by light microscopy. It means that e.g. genotypes

taxonomic groups. Therefore, we designed a (hope- may be hidden among certain morphotypes. A typ-

fully) user-friendly guide for ecologists to unravel ical example is the species complex of Neobodo

the “HNF” black box. In contrast to the few already designis (Scheckenbach et al. 2006). The guide

existing taxonomic keys/reviews (e.g. Bass et al. comprises all the main groups, so that a large num-

2009; Cavalier-Smith and Chao 2010; Lee et al. ber of species morphotypes can be determined at

2000; Patterson and Hedley 1992; Patterson and least to the level of genus. Special attention was

Larsen 1991; Zhukov 1993), the present guide is given to the most interesting and ecologically rel-

compact and focuses on the dominant and common evant forms. The taxonomic categories were used

forms of HF occurring in pelagic and benthic fresh- according to the recently revised classifications by

water habitats. Following the idea of Foissner and Adl et al. (2012).

Berger (1996) with regard to , we designed Within the next decade, next generation

a guide which should be useful also for biolo- sequencing of field samples will give more detailed

gists not specialized in the study of heterotrophic insights into the structure of HF field communities

844 A. Jeuck and H. Arndt

Protists

With 1 or 2 flagella With Without flagella, but with Without flagella/cilia, but Without flagella/cilia, Without

(=( relativelyl ti l llong cilia more numerous relatively short with rigid star-shaped but capable of flagella/cilia, but

comparedto the cell size)size). than 2 cilia for locomotion and/or arrangedaxopods producing with plastids

Cell size ranges from less flagella ingestion (=, stiffened pseudopodia for (photosynthetic)

than 15μm to more than by microtubules) or ingestion and

50μm. Colonies and tentacles locomotion

aggre gations may occur.

Flagellates CChart 6 CCiliates Heliozoans, Amoebae

suctorianssucto a s

Not considered here

Not to confuse with

other very small and

often free-moving

Autotrophic; Heterotrophic; organisms which are

compact and clearly chloroplasts not clearly visible, difficult to differentiate between not : e.g.

visible chloroplast ingested and functional chloroplast; groups containing also large bacteria, and

mixotrophic or autotrophic forms are asterisked ( ) als o metazoans (e.g. t urbellarians,

rotifers)

S ee literature

referring to

autot trophhiic

flagellates Fas t-tt rack to the main groups of heterotrophic flagellates

Chart 8 & 9 Chart 4 Chart 2 Chart 5 Chart 12 Chart 12 Chart 10

Important

characterh t

Choanoflagellates Chrysomonads Ciliophryids Bicosoecids Euglenids Euglenids Free-living

Monosiga Spumella Pteridomonas Bicosoeca Entosiphon Petalomonas kinetoplastids Neobodo

Cryptophyceans Thaumatomonads Apusomonads Cercomonads Spironemids Diplomonads

Goniomonas Gyrodinium Apusom onas Cercomonas Spironema Hexamita

Chart 14 Chart 3 Chart 15 Chart 13 Chart 11 Chart 6 Chart 6

Chart 1. User-friendly guide to common heterotrophic freshwater flagellates. All scale bars in the guide indicate

10 ␮m.

Guide to Free-living Heterotrophic Freshwater Flagellates 845

Heterotrophic p flagellg ates

SinglSinglee cells Colonial,Colonial cellcellsso oftften

liv ingg in a consortium CharChartt7 7

At leasleastt 1 flagellumflagellum visvisiibleble MoreMore than 2 flagellflagellaa visible

2 flagellfl lla (sometimesti onlyl 1 visiblei ibl ) 1 fla gellum visible Chart 6

PreP sence off collarll

Chart 3

With coll ar, funnel-shaped (use phase contrast); the Without collar ((use phase contrast))

createst a filterfilt current;t prottoplastl t (without( ith t theth collar)ll )

< 20 μm F lagellum gliding or non

glidinlidig; presence of f

tentacles CellC ll bbodyd

Choanoflagellates Ciliophryids

oofteftenn

Collar shape varies; collar Tentacles appear

spherical;

consistsi t off a corona off as a collarll unded r Phalansterium

flagellumg 3-5

microvilli; pprotrotooplastplast normallnormallyy the microscope

Cienkowski 1870

times longer

egg-g shapeshapedd or sphericherical;al;

than cell; cell

commonlyy sessile ((attached

This Chart Amoebozoans

upp to 17μm17 m

to the substrate), some free-

swimmin g

GliG lidindingg flaflagellumgellum;; no tenttentacles NoNonn glidglidinging flagellum;flagellum; witwith/withouh/without tentacles

Chart 8

Cell shape/length Cell shape/length/with or without tentacles

PosteriorP t i endd FlFlagellumll (re( llativelyti l SeS condd glilididing flafl gellumll LLeaf-f WithWith ttenttacles,l sometimesti WithoutWith t ttentacles,t l

of ththee cellcell bodbodyy thick)thick) rigid while sometimes ununddeerr cell bodbodyy shapehapedd cell with oopticalptical refractingrefracting somsomee with a

is turned glidinggliding exceexceptpt frofromm the anandd hardhard to ssee;ee; originatinoriginatingg body;body; granulegranules,s sometimes plasmatiplasmaticc stalk at the

upwardsp at a tipp;; dorsoventrally from a prp oboscis edgesg of in one ringg around the pposterior end;; manyy

sharp angle; up flattened or metabolic (=trunk-like protrusion of the cell flagellum; some with mixotrophic forms;

to 10μm (=changeable cell the cell) at the anterior end curled up; a stalk at the posterior second flagellum

Common species Chart 12 cell length Chart 4

Glissomonads

somet imes capable of this, not visible using light microscopy),

F ree-living kinetoplastids Vørs 1992 Chart 10

Chart 2.

846 A. Jeuck and H. Arndt

2 flagella

(second flagel lum not always visible)

Without lorica With lorica (shell-like

covering)

CharChartt5 5

NonNon gglidingliding Gliding (som(somee species (e.(e.g.g. Bodo saltans)s) also

tempo rarily attached to substrate); 1 anterior

flagellum and 1 gliding flagellum; heterodynamic:

Chart 4 anterioriflllb flagellum beats, glidinglidi flagellum fl ll trailsil

Flagell lla //cell ll sh hape/m /ovement t/l/length;th position itiffl of flagella;ll changeabilityhbilit offllh cell shape;

capablapableeorinc or incaapablpableeofpr of producininggp psseudopodieudopodia

Flagella originate ventrally to FlagellaFl ll circai equall FlagellFlagellaa insinsertingerting subapically Posterior edge edge of RlRelativelyti l thithickk SSecond glidinglidi ffllagellumll

slightlyli htl subapicallb i lly iin a in lengthlength;; ccapableapable of in groove; usuallyusually wiwitth tthhee cell body body is flagella,flagella originateate sometimessometimesunder under cell

longitlongitudinalud inal groovegroove (hard(hard to prproducinoducingg acronem (!)( ) ((=diminution at movingg upp and apicallyapically in bodybody andand hardhard to see;see;

seesee),) beatinbeatingg to the lefleftt anandd ppseudopodiap ; the end of the flagella); cell down; slightly emarging ation anterior flagellumg lies in

rightg constantly;y; egggg-shapedp strong metabolic body is oval, ellipsoidal, egg- metabolic (=depression); a proboscis (=trunk-like

to l eaf-shaped and partially (=changeable cell shhapedd or bbean-shaped;h d (=ch( hangeablble cellll cell body egg- protrusion of the cell);

flattened , partially with shape);h ) amoeboib idd; relativelrelativelyy constant cell shape,shape shape);pe); < 8μm shhdtaped to mettabolicb li ((=chhangeablble

verrucous surff ace; postterioi r 5-5 60μm slightly metabolic ellipsoidalellipsoidal and cellcell shashape)pe) andand somesome

enendd lifted;lifted; capablecapable of ((=changeablg e cell shapep ); flattened; rigid produoducece pseudopodia;

produ ciningg pspseudopodieudopodiaa (nonott usually < 10μm cell shapep ; upp to 10μmμ

alwaysy visible);); 10 - 25μmμ 6-100μm

Cercomonas Free-livin g Capable or incapable

DDujardin j di184 1 kinetki topllastidstid off producingd i

FFlagellalagella lengthlength ps eudopodia; with oror

Cercomonads without amoebaa-like Euglenids Apusomonads

ChartChart 10 movements;; with or

without emargination CChart a t 12 CCharta t 133

FlFlagellall equal FlagellaFl ll diffdifferentt CharCh t 11 (=depression)

in length in length

CCommon genera examplel s:

Partlytly leaf- Egg-Egg shapedshaped cellcell Leaf-f shapedshaped cecellll body,body EEgggg -shapedshaped to Pseudopodia Incapacapablb e ofo prp oduoducingg

shapehapeddce cellll body; withwith scales ovaovallor or pointepointeddat at ththee sphericasphericall anandd posteriorly alonalonggthe the ps eudopodia; with emargination

body;y; without on the cell pposterior end;; without flattened cell glidingg g flagellum;g ; (=depression; at the flagellar

scales on the surface (visible scales on the cell surface, body; without am oeboid movements; insertioni ti alongl theth edged (“nos(“e ”))”));

cell surface; using high complex life-cycle with scales on the withou t emargination anteriorly depresseddepressed, lalaterallterallyy

ththreaddliklike magnificationifi ti ) thickthi k cystt walls;ll antert iori cellll surfacef (=depression( d i ) compressed; anterior flagellumtm thin,

ps eudopodieudopodiaa flagellumflagellum typicallytypically waving often difficult to see

mamayy be formedformed

Gyromitus Thaumatomastix ThaumatomonasProtaspis Bodomorpha Ancyr omonas

Skuja 1939 Lauterborn 1899 De Saedeleer 1931 Skuja 1939 Hollande 1942 Kent 1880

ThThaumatomonadst d ThThaumatomonadst d ThThaumatomonadst d CCryomonadds GlGlissomonadsi d AAncyromonadds

Comm on species examples: Common species examples:

G. disomatus T. setifera T. lauterborni P. verrucosa B. reniformis B. minima Ancyromonasy

Skuja 1939 Lauterborn 1899 De Saedeleer 1931 Larsen & Zhukov 1978 (Hollande 1942) Kent 1880

Patterson 1990 Hollande 1952

Chart 3.

Guide to Free-living Heterotrophic Freshwater Flagellates 847

Non gliding

Flagella length/position; presence of refracting particles/stalk/grooves; cell length

Flagella different in length (longer Flagella equal or different in 2 flagella equal or different in 1 equatorial (usually hard to

flagellum somet imes up to five length; cell attached by one length; cells always in see) and 1 longitudinal

times longer than the cell body; flagellum to the substrate; suspension; with refracting flagellum; clearly

short er flagellum often hard to see); actively swimming (rotating); particles (ejectisomes; only distinguishable from all other

if attached, then by a plasmatic very tiny cell, 1.5-4μm visible using high flagellates by 1 longitudinal

stalk (never attached by the magnification with phase and 1 equatorial groove;

flagellum=different to bicosoecids;; contrast); anterior end laterally ca. 8-ca.400μm

2-30μm flattened; 5-40μm

Chrysomonads Naked bicosoecids Cryptophyceans Dinoflagellates

Chart 14 Chart 15

Common gro up, several not yet described

species, often only dist inguishable by

molecular techniques (Arndt et al., in prep.)

Occurring benthically; with plastids: 2 green, gold-

colour ed, sometimes olive-green, red or blue plastids

(only visible using high magnification with phase contrast);

cor tex slightly metabolic (=changeable cell shape)

Presence of scales

(usually only visible using phase contrast)

Without scales With scales

Mode of nutrition

Heterotrophic Mixotrophic (slightly greenish or yellowish

chloropl asts are often visible)

Spumella Ochromonas Paraphys omonas

Cienkowski 1870 Wyssotzki 1887 De Saedeleer 1929

Common species examples:

Co mmon species examples:

Single cells of Scales

An thophysa and

Siderodendron and Schematic drawing of a

other genera scale as visible under the

(usually colonial) elect ron microscope

are difficult to

Variable

dist inguish from

iin siize

Spumella

Spumella vulgaris Ochromonas oligochrysis Paraphys omonas vestita

Cienkowski 1870 Ettl 1978 (Stokes) De Saedeleer 1930

Occurring pelagically and benthically; some living in colonies;

some species with lorica;; sometimes attached to the substrate by

a plasmatic stalk (or( exopolymeric matrix) at the posterior end

Chart 4.

848 A. Jeuck and H. Arndt

With lorica

(sh ell-like housing, often only

visiblei ibl usingi phah se contrt astt))

Not attached to the lorica by the flagellum, Attached to the lorica by the posterior flagellum (sometimes attached by plasmatic thread)

Lorica shape; flagella; lorica length Lorica shapeape;; flagellaflagella;; lorica length

CoCommommonn gengenereraa examexamplples:es:

Loricav vereryy thinthin;1; 1 Cone-shape shapedd lorica very Lorica a attachedttachedby by Cone-shapedp and pointedp Lorica lateralllaterallyy compressed,compressed

longg and 1 short thin; cell bodyy with stalk to substrate; cell lorica; second flagellum rounded at the bottom,, ppointed,,

emergent flagellum; chromatophore (); body attached to lorica hard to see (free- ending in a thin stalk; second

10-15μm 1 long and 1 short emergent by plasmatic thread; swimmi ing specimens:i flagellum often lying in a ventral

flagelgellum;l m sometimes 1 llongong andand 1 shortshort clearlarlyy distinctdistinct fla gellum); groove; cellll nott formingf i a largel

ps eudopodia; 7-20μm emergent flagellum;flagellum; celcelll forminformingg a larglargee lilipp to lilippto to ononee side side;; 8- 8 12μm

55-28μ m one side;;μ 10-15μm

CalycomonasCalycomonas Poteriochromonas StokesiellStokesiellaa Histiona ReclinoReclinomonasmonas

Lohmanmannn 19081908 ScherffeScherffel 1901 LemmermanLemmermannn 1910 VoigtVoigt 19190202 FlavFlavinin & NeradNerad 19199393

Chrysomonads Chrysomonads Chrysomonads Jakobids

Bicosoecid s

Common species examples:

Aqueous lorica, Bell-shaped lorica; cytostomal Bell-shaped lorica; cytostomal Ellip soidal, egg-shaped to Cylindrical lorica;

transparent;transparent 7-7 1010μm areaarea usuallys all slightlyslightl narrowenarro edd; areareaa stronglystrongl enlargeenlarged;d vase-shapedh d lloricai ; flflagellumll upp to caca. 4040μmm

posterioposteriorr end button-like posterior end roundeundedd anandd is been unreeledunreeled in a jerkingjerking

thickenedickened;; 12-12 14μm poinpointteded slighslightlytly; 2020-40μ m manne r (cf(cf.. tongunguee of a

chameleo n)); ca. 30μmμ

Bicosoeca paropsisp p Bicosoeca pplanctonica Bicosoeca campanulatp a Bicosoeca lacustris Bicosoeca cyly indrica

Skuja 1956 Kisselew 1931 (=B. crystallina Skuja James-Clark 1867 (Lackey) Bourrelly

1956) 1951

(L(Lack key) BBourrellyll 1951

Other commo n species examples:

B. lacustris

mighti ht alsol fform

colonies, see

CharChartt 7

B. exilis Penard 1921 B. conica Lemmermann 1912 B. kepneri Reynolds 1927 B. ovata Lemmermann 1914

Chart 5.

Guide to Free-living Heterotrophic Freshwater Flagellates 849

More than 2

flagella Number of flagella

Number of flagella; cell shape/length Other number of flagella, 4 flagella 2-162 flagella rotation- symmetric Egg-shaped to ellipsoid; Spherical, cells often covered 2-16 flagella (asynchronous), Clubbed, drop-shaped or comma-

20-30μm by a layer of mucus; 8-20μm star-shaped inserting; 5-20μm shaped; obligate anaerobic; 8-30μm

Collodictyon Quadricilia Multicilia lacustris Tri mastix

Carter 1865 (Skuja) Vørs 1992 Cienkowsky 1881 Kent 1880

Collodictyonids Protista incertae sedis Amoebozoans Preaxostyla

Number of flagella; cell shape/length

12-24 flagella; Many short flagella (asynchronous), all on tip 2 rows of 12 flagella; egg-shaped with a slightly

ca. 20μm at the cytostomal area; conical cell; 10-20μm conical posterior and a truncated anterior end;

obligate anaerobic; 14-20μm

Paramastix conifera Spironema multiciliatum Hemimastix

Skuja 1948 Klebs 1893 Foissner, Blatterer & Foissner 1988

Pr otista incertae sedis Spironemids Spironemids

2 distinct, posterior flagellaPresence of posterior flagella Without posterior flagella

CelCelll shape; fflagella/celllagella/cell lenlenggthth Cell shape; number of flagella; cell length

Irregular posterior cell projection; 6 Oval, spindle-shaped, cigar-shh apedd Almost triangular, spindle- Egg-shaped or pear-

anterior flagella slightly shorter than cell or flattened; 6 anterior flagella circa shaped or spiral; 6 flagella; shaped, flattened; 8 flagella;

body; obligate anaerobic; 16-22μm as long as cell body; 10-35μm obligate anaerobic; 12-35μm obligate anaerobic; 7-30μm

Urophagus Hexamita Trigonomonas Trepomonas

Klebs 1892 Dujardin 1838 Klebs 1892 Dujardin 1841

DiDipllomonadsd DiDipl lomonadsd Diplomonads DiDiplomonadsl d

Chart 6.

850 A. Jeuck and H. Arndt

Colonial

PfPresence of collar;ll numberbffl of flagellall ; presencepresenceof of lorica( (housing);housing); presenceence of

stalk

Without collar; 1 visible flagellum Without collar; 2 flagella; With collar; 1 flagellum; WithoutWithout collar;collar;2 2 flaflagellgellaa (secondnd

(second(secondsedto used to attachattachto to thethe lorica)); withoithout lorica; withoithout t sta lk withith or witho ithout t stastalklk flagelluflagellumm hardhard to see);see); withowithouutt loricalorica;;

witwithhor or without sta lk witwithh stalkstalk

FForm off ththe colonl y; FormF off ththe colonyl FormF off ththe colonyl FormF off ththe colonyl

with or without stalk

ColonieColoniess star-shstar shaped: Colonies shapeapedd ColonieColoniess first ColonieColoniess branched or ColonieColoniess branched or Colonies

6-30 cells attached byy like a tree, branched sshapehaped like a unbranched, cells unbranched, cells not branched;

the bottom of the and stuck together cushion or attached or not attached attached to a stalk, young

lorica to each other; (up to five- s ponge-like, to a stalk, some attached colonies disc-shaped to colonies are

without stalkstalk ( freefree- storey ed);ed); withwith long older colonies by inintercellulartercellular bbridgesridges to sphericasphericall or tutubulabular,r colourlesscolourless,

swimming) stalkstalk ( attached) oftften llobbatet eaeacchh other,her otheotherr cellcellss finger-shapeapedd to linear; oldolderer onesones

radially,s, sppongongee-like like or flagellugellumm 2-32 3 timestimes oofteftenn

tu belike arranged longer than cell bodies brownish

see C harhartt 9: Colonial

choanoflagellates

BiBicosoeca llacustrit is BiBicosoeca petiolatai l SSpongomonas PhPhalansteriul i m

Jamess-ClarClarkk 18671867 (Stein) Bourrelly 1951 SStein tein 18187878 Cienkowski 1870

Bicosoecids Bicosoecids Spongomonadsp g Amoebozoans

Withhou t CCommon speciesi examplel s:

stalkstalk seseee aalsolso Phalansterium

Chart 5 digitatum

SStein tein 18187878

Number of cells per

Spongomonas uvella

SStete in 18187878

Bico soeca ‘socialis‘=colony

form of Bicosoeca lacustris

Co mmon species examples:

Common species example:

Cluster of up to 8 Number of cells often

cells or single < 60, spherical or

cells;ll iirregularlyl l hhemisp i hhericali l

branched systsystemem colonies; umbel-likelike

of rigrigidid stalks witwithh a branched stastalklk

Cells also free-

swimmin g; if

SiderodendronSid d d AnthophysaA th h withith redd stigti ma

Pringsheim 1946 Bory 1822 (eyespot) A. steini Senn

Chrysomonadsy Chrysomonadsy 1900

Siderodendron manganiferumg Anthophysap y vegetang s

Pringsheim 1946 (Müller) Stein 1878

Sin gle cells are difficult to distinguish from Spumella – like flagellates

Chart 7.

Guide to Free-living Heterotrophic Freshwater Flagellates 851

Choanoflagellatesare polymorphic species which build different life-cycle-forms (see Chart 9)

Choanoflageg(p)llates (Craspedida)

PresencPresenceeof of

(y,g(=membranous layer, enclosing the cell body)y) Without theca With theca Number of collarscollars,, presence of stalk

One collar Two collars One collar;; stalked

MiMonosiga Kt1880Kent 1880 DiDiplosiga l i Fl1892F renzel 1892 CdCodosiga i Jamess-ClarClarkk1868 1868

Common o ssppeecceseies examples a pes:

Monosig a angustata

Kent 1880

Monosigsiga a ovata

Kent 1880

D iplosiga socialis

ColonColonyy formform of Codosiga: FFrenzell 18189292

Monosiga fusiformis see next Chart

KKent 1880

Latest investigations showed: Depending on life-cycle, several species might form swarmers easily mistaken for Monosiga – like forms.

ForFormm of ththee theca

Cupp-shaped p theca Flask-shapedp theca Tube-shapedp theca

Non structured theca Struct ured theca (w( ith spikep s))

Salpingoeg ca Salpingoeca Lagenoeg ca Salpingoeg ca Aulomonas

James-Clark 1868 James-Clark 1868 Kent 1880 James-Clark 1868 Lackey 1942

Co mmon species examples:

This might be the

Common sin gle cell of the Type species species: colonylfi forming examplp e: Stelexomonas dichotoma (see

Chart 9)

S. schilleri S. amphoridium Lagenoeca poculiformis S. cylindrica Aulomonas purdyi

(Schilhillerl ) StStarmach 1968 JJames-ClClarkk 18186688 ScS hillhiller 19531953 FFott tt 19531953 LLackeyk 19419422

S. urceolata S. oblonga S. vaginicola S. gracilis S. frequentissima S. globulosa S. variabilis (=Lagenoeca

Kent 1880 Stein 1868 Stein 1878 James-Clark 1868 (Zacharias) Lemmermann 1913 Zhukov 1978 variabilis) Skuja 1956

Chart 8.

852 A. Jeuck and H. Arndt

Life-cycl y e of choanoflagellateg s,, exampleforSalpingoeca (after Dayelet al. 2011)

CColonial choano fflagellates (C(Craspedida) )

Form of colony

Unbranc hed colonies Branched colonies

SittingSitting separatelyseparately or in Usually 44-15 cellcellss sitting togethetherr MMany stalkedt lk d cellsll radiallydi ll 6-6 60 cellscells maymay be

groupsgroups at tthehe ttiipp of thethe stalk,alk, liklikee a pearpearll necklace;necklace; singlsinglee cells aarrangedrr anged in a ggelatinouselatinous heldheld together by a

stalk is 2-10 times longeg r attached byy intercellular bridges,g matrix gelatinousg cluster

than cell body free-swimming

Codosiga Desmarella-lik e forms Sphaeroeca-like forms Proterospongia-like forms of

JJames-ClClarkk 18186688 off choanoflagellateh fl ll t s off choanoflagellateh fl ll t s choanoflagellateh fl ll t s

Desmarella/ Sphaeroeca/Proterospongia – like forms might be life-cycle forms of other species

CCo mmon speciesi

example:

SingleSi l cellll

If occurring as single cells (detached from the colony),

Codosiga botrytis those are ca pable of producing pseudopodia at the

(Eh(Ehrenberg) b ) KentK t 1880 posttierior end d.

Common sp eciesies examplexample:

2 to severalseveral cells,cells atattachetached by CellCellss not attachedttached to a

lonlongg and thinthin individ uualal stalks to stalk, surrounded by a

a common stalk;; branched like dichotomous tube of

an umbel or bifurcated organiic matter

CodosigaC d i Stelexomonas dichotoma

James-ClarkClar k 18681868 Lackey 1942

Accor ding to latest molecular investigations,

CodosigCodosigaa umbellata

this morphotype is present in several cryptic

SStetein 18187878

speciesi whichhi h currentlytl migi ht nott yett beb

distingu ished

Chart 9.

Guide to Free-living Heterotrophic Freshwater Flagellates 853

Fre e-living kinetoplastids

2 flagella; agitated 1 visible flagellum and 1

Numb er of flagella; movement; position of the ingestion apparatus

swimming; ingestion moveable anterior proboscis

apparatus at the tip of the („nose“ =trunk-like

anterior end, partially (e.g. protrusion of the cell) (with

Rhynchomonas nasuta (Stokes 1888)

Bodo saltans) attached to smaller flagellum); proboscis

– species complex substrate beating frequently; ingestion

Neobodonids apparatus located in proboscis

Movement

Common species examples:

Cell body swings abruptly around the base

Anterior flagellum is swinging Fast and equal swimmer;

Anterior flagellum

of the flagellum; cell is ca. 5-10μm distant

around the anterior part of the when crawling on the

‚paddles‘ and

from the substrate, attachment by the tip of

cell like a lasso; attachment by substrate, then cell is

appears hook - like

the recurrent flagellum; anterior flagellum

the recurrent flagellum is trembling and swinging;

creates a whirl to transport the bacteria into

frequently changing browsing the surface with

the ingestion apparatus

the anterior flagellum

Bodo saltans Neobodo designis Neobodo curvifilus Parabodo

Ehrenberg 1832 - species complex (Skuja) Vickerman 2004 - species complex Griessmann 1913 Skuja 1939

Eubodonids Neobodonids Neobodonids Parabodonids

Commo n species

example:

Other common genera examples of neobodonids:

Dimas tigella Rhynchobodo

Parabodo nitrophilus Lackey 1940 Sandon 1928 Skuja 1939

Chart 10.

(e.g. Pawlowski et al. 2011, Stoeck et al. 2010, knowledge of HF (recommended literature: e.g.

and Domonell et al., unpubl. observ.). However, Hausmann et al. 2003; Leadbeater and Green

their application still bears a lot of problems 2000; Patterson and Larsen 1991). The systematic

concerning the following points: 1) many specific position of flagellate groups considered in the guide

primers are necessary to cover the whole HF is given in Figure 1 following the recent revision by

diversity due to the wide molecular diversity of Adl et al. (2012). Following the idea of Foissner and

HF (some groups will be overlooked), 2) active Berger (1996) with regard to ciliates, we designed a

and inactive forms are difficult to differentiate, 3) polytomous guide. The first Chart contains a gen-

databases are incomplete (Will and Rubinoff 2004) eral overview of the most important characters of

and contain high numbers of errors (Prosdocimi the main groups of protists. The user is led to a

et al. 2013). Quantitative morphotype studies will “fast-track” regarding the main groups of HF (Charts

provide helpful information to increase reliability 1–15) by schematic drawings with arrows point-

of databases and offer important knowledge on ing to the important characters. If identification is

the ecology and evolution of HF groups significant already sufficient the user is led to the special

in ecosystems (Pawlowski et al. 2012). We would Chart of the organism. If not, he should proceed

like to increase the resolution of these quantitative to Chart 2. The user is led from obvious characters

morphotype studies by this taxonomic guide. (e.g. colonial or single cells, number of flagella) to

more detailed descriptions. Features of organisms

which are used to discriminate between different

taxa are set within boxes while the discriminator for

How to Use the Guide

the subsequent path of identification is shown with-

The present guide is intended to help also unex- out boxes. Figures are added to the descriptions of

perienced researchers to identify HF morphotypes, characters to accelerate and increase the chance

although it is recommended to have a general of identification. All scale bars in the guide indicate

854 A. Jeuck and H. Arndt

Cercomonads Ultrastructural and

molecularl l analysil is isi

often needed for a

Cell/psp eudopodiap shapep /lenggth;; flagellag movement/lenggth;; changeabilityg y of cell shapep distinct differentat ion

Comp rising small, but also largest known Small and compact cell body, Often smaller cell body; various Spherical or oval cell body;

cerc omonads; pseudopodia mostly when moving, then spindle- forms of pseudopodia; anterior various forms of pseudopodia;

rounde d or finger-like;- often with long shaped; short flagella, flagellum is slowly rowing, very long anterior flagellum, flagella, posterior trailing; slightly posterior trailing;5-15μm posterior trailing;3-18μm posterioroftennotattachedto

metabolic; 5-60μm cell body; ca. 10-13μm

Cercomonas Eocercomonas Paracercomonas Nucleoce rcomonas

Dujardin 1841 emend. Karpov et al. 2006 Karpov et al. 2006 Karpov et al. 2006 Arndt et al. 2012

Co mmon species examples: Other

forms

Cercomonas plasmodialis Eocercomonas ramosa Paracercomonas metabolica Nucleoce rcomonas praelonga

Mylnikov 1985 Karpov et al. 2006 (Zhukov and Mylnikov 1987) Brabender et al. 2012

Karpov et al. 2006

Ovoid cell body, dorsoventrally Highly and fast varying cell shape; various forms of Oval or drop-like cell body; various forms of

compressed; rigid cell shape; pseudopodiaeudopod a ana ywhere e oon tthee cell;ce ; aanterite oor flagellage um ps eudopodia; very short anterior flagellum,

3-9μm beats very often; fast gliding cell; extremely metabolic; not t apering, directed to the left, posterior is

9-13μm longe r; ca. 5-8μm

Cav ernomonas Metabolomonas Brevimastigomonas

Vi ckerman 2009 Kiss et al. 2012 Nitsche et al. 2012

For detailed descriptions of these genera see: Bass et al. 2009; Brabender et al. 2012; Karpov et al. 2006

Chart 11.

␮

10 ␮m. The guide leads only to the species or genus 5-20 l on a prepared microscope slide or an Utermöhl cham-

ber (Utermöhl 1958; HydroBios GmbH, Kiel, Germany; Fig. 2).

morphotype. It is widely accepted that many pro-

The HF composition has to be analysed within one hour after

tistan morphotypes contain a few up to hundreds

sampling by means of a phase contrast microscope equipped

of genotypes (typical examples are the common

with high resolution video-recording which is helpful for further

species Neobodo designis (Scheckenbach et al. and later identification. 20x, 40x objectives are helpful for rough

2006) or Codosiga botrytis (Stoupin et al. 2012)), morphological observations and quantitative counts. Individual

samples can be analysed within a few minutes (particularly

see above. If video sequences (Supplementary

critical for pelagic samples). The use of a 63x long distance

Material) are available for the organisms, they are

objective or water immersion objectives (63x and 100x; with a

marked by a video icon ( ). Some additional lit-

long working distance) are recommended for morphotype iden-

erature is given on the Charts (Cavalier-Smith and tifications. Phase contrast equipment is mostly indispensible to

Chao 2010; Bass et al. 2009; Brabender et al. 2012; analyse position and movement of flagella, the presence of col-

lar, lorica, or stalk of some flagellates. Differential interference

Karpov et al. 2006). Due to the fact that major forms

contrast (DIC) equipment might additionally help to identify cell

of heterotrophic flagellates are ubiquitous in marine

structures such as paramylon, ingestion apparatus, or

and freshwater environments, this guide may also .

be useful in parts for an orientation regarding flag- A realistic compromise should be a combination of counts by

the living-droplet method with counts of chemically fixed sam-

ellate groups in brackish water systems.

ples and analysis by epifluorescence microscopy. The following

fixatives are commonly in use: 2% glutaraldehyde (e.g. Caron

1983; Choi and Stoecker 1989), 2% formaldehyde (e.g. Porter

Methods and Feig 1980; Sherr et al. 1989), buffered formaldehyde (e.g.

Børsheim and Bratbak 1987; Sherr and Sherr 1983) and 0.5%

The guide is based on an analysis using the classical living- acidic Lugol’ solution (10 g I2, 20 g KI, 10 g sodium acetate in

droplet method by phase contrast/DIC light microscopy (e.g. 140 ml aq. dest.) plus 3% formaldehyde (e.g. Sherr et al. 1989).

◦

Arndt and Mathes 1991; Gasol 1993; Massana and Güde The fixed samples need to be kept at 4 C in the dark

1991). This method is reliable to detect morphological and until staining. Fluorescent dyes (e.g. DAPI (= 4 6-diamidino-

behavioural characteristics of even very small HF regarding 2-phenylindole, Sigma Lot 47H4107, Porter and Feig 1980)

their main taxonomic groups – in contrast to hidden charac- are suitable for staining. After staining the sample should

␮

teristics within samples treated by fixatives. Pelagic flagellates be filtered onto a 0.2- m membrane filter using vacuum not

(undiluted samples) and benthic flagellates (diluted by a fac- exceeding 150 mm Hg. For an even distribution of the parti-

␮

tor of 5- >20 with filtered water) can be studied in droplets of cles, another membrane (0.6 m pore size) may be placed

Guide to Free-living Heterotrophic Freshwater Flagellates 855

Euglg enids

1 visible flagellum; second flagellum reduced or fused to 2 flagella,g , different in lenggth,, heterodynamiy c (the flagellg a

the side of the cell;; storageg granuleg ((pparamylony ) ppresent have different beating patterns)

Changeability of the cell shape

Metabolic ((=chchangeable celcelll shape) RelativelRelativelyy rigid

Form/movement of flagellug m/cell Form/movement of flagellum/cellg body;

body; cell shape/length cell shape/length

CoCommommonn generagenera examexamplples:es: CommoCommonn generagenera examples:examples:

Well developedevelopedd Relatively thick flagelluflagellumm, Relatively thick flagellum, Flagellum stistiffedffed in frontfront FlagelluFlagellumm most aactivective

flagellum, most active beatating ing in whiplash fasfashiohion; beats in a lashingg manner; of the cell, most active near the tip;p front end

near the tip; cell body cell body actively swimming sickle-shaped or bean- and vibrating near the of the cell enlarged to

squirms actively; (rotating) or metabolically shaped, laterally or tip; egg-shaped, ‘flickering’ funnel-

cigar-, cone-shaped gliding ; spindle-shaped to ddorsoventrt allyll flattttenedd; spindlee-shapeshaped,d ovaovall to shhaped fronf tall region,i

to sphericasphericall (often(often cylind ricalrical;; 16 - 80μm 14-100μm triangulartriangular;; rigidrigid cells and ofoftetenn striated;

deformingdeforming),) oftenoften dorsoventrally flattenedened;; 25 - 60μm

flattened; ; 20 -100μmμ 6-50μm

PeranemPeranemaa Astasia Menoidium P etalomonas Urceolus

DujardiDujardinn 1841 DujardiDujardinn 1841 PerPerttyy 1852 SStein tein 18185959 Mereschkowskschkowskyy 1879

ConfusioConfusionn withwith

Notosolenus ppossible:

obs erve carefully if

P

there is a second

Paramylon (P)

P flf lagellumll (when( h tthhe cellcell turns) P

P

ConfusioConfusionn withwith

Heteronema possible:

observe carefully if there Ingestiong apppparatus; form/movement of flagella;g changeabilityg y of

isi a secondd flagellumfl ll cell s hape; cell length; presence of storage granule (paramylon)

(when the cellcell turns)

SomeSome common genereraa examples:

With ingestioningestion apparatusapparatus (often(often hardhard to With ingestioingestionn apparatusapparatus With ingestioingestionn apappaparatus WithWith ingestioingestionn apapparatuparatuss

see); swimminingg anteanteriorrior flagellum flagellum weakerweaker (small)(small);; swimminingg anterior (often(often hard to see);see); swimming (wel(welll developedeveloped anandd

and often shorter than posterior,p , beatingg flagellumg (tip( p in tail motion)) ante rior flagellumg (most( active visible);); postp erior

normally; posterior trailing, contracts to jerk often longer than trailing near the tip) longer than flagellum trailing behind

the cell backwards; cells dorsoventrally posterior; often metabolic trailing posterior; cells t he cell when moving;

flattttenedd; slighli htltly mettabob licli (=ch( hangeablebl (=ch( hangeablble cellll shah pe);) ddorsoventrt allyll flattttened;d rigid;i id cellsll ddo rsoventrallyll

cell shape)shape) or rigid; 14-100 μm 25-25 100μm 1010-30μm flattenedttened; ; rigid; 10 -30μm30μm

Anisonema Dujardin 1841 Heteronema Dujardin 1841 Notosolenus Stokes 1884, Entosiphon Stein 1878

em. SkSkuja j 11993399

Ingestion

ConfusioConfusionn withwith ConfusioConfusionn with apparatust

Per anema possible Petalomonas P

if the second possible if the

flagellum is second flagellum is

ove rlooked overlooked

P

Second flagellg um

Chart 12.

856 A. Jeuck and H. Arndt

Apusomonads

Position of the anterior and recurrent flagellum; changeability of the cell shape

Anterior flagellum lies in a proboscis (=trunk-likeprotrusion of the Anterior trailing flagellumprotrudes from the proboscis (=trunk-like

cell); recurrent flagellum often hard to see; cells are metabolic protrusion of the cell); recurrent flagellum lies directed pyposteriorly under

(=changeabl e cell shape) and some produce pseudopodia the cell; flattened cell body is slightly metabolic (=changeable cell shape)

Apusomonas

Alexeieff 1924 Common species example:

Easily

confused with Apusomonas proboscidea

Rhynchomonas Al exeieff 1924

nasuta!

Amastigomonas Manchomonas Podomonas Thecamonas Multimonas

De Saedeleer 1931 Cavalier-Smith 2010 Cavalier-Smith 2010 Larsen & Patterson 1990 Cavalier-Smith 2010

For detailed descriptions of these genera see: Cavalier-Smith and Chao 2010

Chart 13.

Cryptophyceans

Flag ella length; presence of dark refracting particles( (=ejectisomes) (partly hard to see); presence of bright

refracting storage granulum; presence of striations; cell shape; mode of movement; cell length

Flage lla approximately equal in length; Flagella approximately equal in length; ejectisomes Flagella different in length; ejectisomes

ejectisomes orientated in a row around the are clustered around a emargination (=depression) scattered within the cell body (only visible

ante rior end of the cell (only visible using high at the anterior end (only visible using high using high magnification with phase

magnification with phase contrast); without magnification with phase contrast); with storage contrast); with storage granulum; without

storage granulum; with 3 - 6 striations; clearly granulum; without striations; circular section; striations; circular section; swimming and

dorsoventr ally flattened; swimming close to the swimming fast in the water column; 15 - 30μm jumping in the water column; 10 -18μm

sub strate; 5 -12μm

Goniomonas Cryptomonas Kathablepharis

(Fresenius) Stein 1878 Ehrenberg 1831 emend. Hoef-Emden & Melkonian 2003 Skuja 1939 Synonym: Cyathomonas Fromentel 1874 Synonym: Chilomonas Ehrenberg 1831

Cryptomonads Kathablepharids

Common species examples: Common species example: Common species example:

Movement:

Similar s pecies fast, rotating in occur in brackish Refracting Many food a waving

ejectisomes

water: vacuoles manner, shorter

e.g. flf lagellum ll bea bt s

G. amphinema around the

Larsen & Cryptomonas paramaecium anterioanteriorr end;end;

Patter son 1990: Ehrenberg 1832 emend. onlyy ppelagicallg y Hoef-Emden & Melkonian occuring. 2003

Kathablepharis

Goniomonas truncata o valis

(Fresenius) Stein 1878 Skuja 1948

Chart 14.

Guide to Free-living Heterotrophic Freshwater Flagellates 857

Dinoflagellates

Cell shape

EggEgg-shaped, ellipsoidal to spherical Other cell shape

PiiPosition of fh the equator ilial groove (cingulum) (il )

OtOtherh positionitif of ththe AtAnterior i positionitif of

cingulum ththee cingulucingulum

CCell ll shah pe; fform off grooves; cellll l lengthth

Common gpgenera examples:

Often with longitudinal Almost bilateral symmetrical; With plates, sometimes also Distinct theca (=membranous

striat ions on the surface; cingulum at the cell’s ornamented with spines, teeth-like layer, enclosing the cell body);

litdillongitudinal groove (sulcus (l)) posteriorti end, dtht the two ends d projjtiections or wings; i cingulum i l almost l t edgedfs of ththe grooves are

parti allallyy spirals to cingulu m, of the cingulu m meet;meet; horizonthorizontallyally, didividesvides thethe bodybody into two clearlclearlyy visiblevisible due to ridges;

ththeetwo two endendssof of ththee cingulum morphologmorphologyych chaangenges duduee to halfs of different length; 20-20 100μm

do not meet;1; 0-50μμm ingestedg food items;; 10- 16-55μμm

50μm

Gyro dinium GymnodiniumPeridiniopsis Peridinium

Kofoid & Swezyy 1921 Stein 1878 Lemmermann 1904 Ehrenbergg 1832

With With

Without

Without chlhloroplal stts chlhlo roplal stts

c hloroplap sts, chloroplasts,

but may but may

contain containt i

coloul redd coloured food food

vacuoles vacuoles

CCo mmon genera examples:l

Cell shappe; positiop n/form of grooveg s; cell lengtg h

Cell body often egg-shaped, usually the

Sometimes egg- or mushroom-shaped cell llongit ituddiinall groove (sulcusl ) extent dsd fromf

body, oftteen dorsoventralldo so e t a y flatteneatte ed;d; 88-50μ50μm the cingulum without torsion; 6-6 50μm

Without With

chloroplap sts c hloroplasts Amphidinium

Katodinium Claparède & Lachhmannmann

FottFott 195719 57 1858/61

Cell shape; positionn/fo/forrmm of grooves;s; cell length

Co mmon genera examples:

With longelongerr or sshorterhorter hornhorn-like Lenss-shapedshaped to round,und almost Elongate,te ellipticalelliptical or lunate,ate

ppr ojectionj s (anter( ior end with Common symmy etrical;; equaq torial grooveg sometimes rounded or

one horn, posterior end with spep cies (cingulum) almost spherical; apiculate, with large spines;

several ), asymmetrically example: longitudinal groove (sulcus) only at 40-450μm

shhaped d; equatoriali l groove ththe posteriort i sideid off ththe cellll bbodyd ;

(cingulum)cingulum ) lieliess horihorizozonntaltally,ly 1616-40μm With

dividedividess thethe cell body intointo two chloroplasts

hal fs of nearlyy eqqual length;g Without

40-450μ m chloroplashl l ts,t butbut may

With contaicontainn

chloroplasts c oloured

CeratiumC ti DiDiplopsall liis CCystodiniumt di i

Ceratium hirundinella food

SchranSchrankk 1791793 BerghBergh 18811881 KlebKlebss 19121912

(O.F. Müller) Dujardin 1841 vacuoles

Chart 15.

858 A. Jeuck and H. Arndt

Figure 2. Schematic drawing of some helpful counting chambers. a) Simple microchamber: Two cover slips

are placed on the slide (each on every end). A 10 ␮l droplet is placed between them and a third cover slip above

the droplet and the two slips. b) Utermöhl chamber for counting living flagellates or sedimented, chemically

fixed samples.

underneath the filter. The filters are generally stored deep-

◦ Appendix A. Supplementary data

frozen at –20 C until examination by an epifluorescence

microscope.

Supplementary data associated with this arti-

It is additionally recommended to establish cultures of HF

cle can be found, in the online version, at

isolated from the samples (e.g. simply by adding autoclaved

wheat grains etc. as a carbon source for food bacteria to parts http://dx.doi.org/10.1016/j.protis.2013.08.003.

of raw samples). This should aid in morphotype identification

of bacterivorous forms and to check whether the determined

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