A Short Guide to Common Heterotrophic Flagellates Of

Home , Apusomonas, Flagellate, Kiitoksia, Lorica (biology), Neobodo, Peranema

Protist, Vol. 164, 842–860, November 2013

http://www.elsevier.de/protis

Published online date 6 November 2013

PROTIST TOOLS

A Short Guide to Common Heterotrophic

Flagellates of Freshwater Habitats Based

on the Morphology of Living Organisms

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. euglenids,

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 mixotrophs (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

Guide to Free-living Heterotrophic Freshwater Flagellates 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 ciliates, 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

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

compared to the cell size). than 2 cilia for locomotion and/or arranged axopods producing with plastids

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

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

50μm. Coloni es and tentacles locomotion

aggregat ions may occur.

Flagellates CChart 6 CCiliates Heliozoans, Amoebae Algae

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 protists: 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

Choanoflage llates Chrysomonads Ciliophryids Bicosoecids Euglenids Euglenids Free-living

Monosiga Spumella Pteridomonas Bicosoeca Entosiphon Petalomonas kinetoplastids

Neobodo

Cryptophyc eans Thaumatomonads Dinoflagellates Apusomonads Cercomonads Spironemids Diplomonads

Goniomonas Thaumatomonas 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 flagell ates

Single cells ColonialColonial, cells oftenoften

livingg in a consortium Chart 7

At least 1 flagellum visible More thanthan 2 flagella visible

2 flagellfl lla (sometimes( ti onlyl 1 visible)i ibl ) 1 flagellum visible Chart 6 PresenceP off collarll Chart 3

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

createst a filterfilt current;t protoplastt l t (witho( ith utt theth collar)ll )

< 20 μm F lagellum gliding or non

glidinlidi g; presence off

CellC ll bbodyd tent acles

Choanoflagellates Ciliophryids

ofteo ftenn

Collar shape varies; collar Tentacles appear

spherical;

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

flagellumg 3-5

microvilli; protoplast normally the microscope

Cienkowski 1870 times longer

egg-shaped or spherical;

than cell; cell

commonlyy sessile ((attached

This Chart Amoebozoans

upp to 1717μmm

to the substrate), some free-

swimmin g

Gliding flagellum; no tentaclestentacles Non gliding flagellum; with/withoutwith/without tetentaclesntacles

Chart 8

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

PosteriorP t i endd Flagellum (relatively Second gliding flagellum LeaL f-f With ttenttacles,l sometimesti Without tentacles,

of the cell body thick) rigid while sometimes under cell body s hapehapedd cell with optical re refractingfracting some with a

is turned gliding except from the and hard to see; originating body;body; granules,granules sometimes plasmaticstalk at the

upwardsp at a tip;p; dorsoventrally from a proboscis 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

shape);h ) 6 - 100μm (=anterior( t i flflagellum);ll ) up tto up tot 1010μm endd (swarmers possible);ibl ) often hard to see;

10μm mixotrophic; egg- to apple- leaf- to apple-

shaped ca. 10 -15μm shaped; ca. 5 -15μm

Some specific EEuglenidsl id ShapeShape of the Phyllo monas contorta

Cercozoans anterioanteriorr Klebs 1892 Ciliophryids Chrysomonads

flagellum;g ;

Common species Chart 12 cell length Protista incertae sedis examples: Chart 4

Common genera examples:

Allantion tachyploon

Sandon 1924

Glissomonads Proboscis ((“nose”) directed Beating proboscis (“nose”)( ) at the anterior

Ciliophrys Pteridomonas

anter iorlyy or more laterally;y end (small( anterior flagellum attached to

Cienkowski 1876 Penard 1889

sometimes capable of this, not visible using light microscopy),

Metopion fluens produ cing pseudopodia; sometimes bulbous at the end of the

Larsen & ca 7- 10μm proboscis; 1 well visible gliding flagellum

Patterson 1990 directdirected ed posteriorly; 3 – ca.77μmμm Metromonads Actinomonas Apusomonads Free-living kinetoplastids Kent 1880 Kiitoksia

Vørs 1992 Chart 13 Chart 10

Protista incertae sedis

Chart 2.

846 A. Jeuck and H. Arndt

2 flagella (second flagellum not always

visible) Without lorica With lorica (shell-like

covering)

Chart 5

Non gliding Gliding (some species (e.g. Bodo saltans)) also

temporarily attached to substrate); 1 anterior

flagellum and 1 gliding flagellum; heterodynamic:

Cha rt 4 anterior flagellum beats, gliding flagellum trails

Flagell lla//cellll shhape/m/ ovementt/l/length;th position of flagella; changeability of cell shape;

capable or incapable of producing pseudopodia

Flagella originate ventrally to FlFlagellall circai equall Flagellainserting subapically Posterioredge of Relatively thick Second gliding flagellum

slightly subapically iin a in length; capable of in groove; usually with thecell body is flagella, originate sometimes under cell

longitudinal groove (hard to producing acronem ((!)) (=diminu( tion at moving up and apically in body and hard to see;

see) beating to the left and ps eudopodia; the end of the flagella); cell down; slightly emargination anterior flagellum lies in

righ t constantly; egg-shaped 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 (=changeable cell cell body egg- protrusion of the cell);

flattened , partially with shape); amoeboid; relatively constant cell shape shape); < 8μm shhapedd tot mettabolicb li ((=chhangeablble

verruco us surface; posterior 55- 60μm slightly metabolic ellipsoidal and cell shape) and some

end lifted; capable of (=changeablg e cell shapep ); flattene d; rigid produce pseudopodia;

producing pseudopodia (not usually < 10μm cell shape; up to 10μm

alway ys visible);); 10 - 25μm 6-100μm

Cercomonas Free-livin g Capable or incapable

Dujardin 1841 kiinetopl t lastidstid of producing

Flagella length pseudopodia; with or

Cer comonads without amoeba-like Euglenids Apusomonads

Cha rt 10 movements;; with or

without emargination ChartC a t 12 CChartt 133 Flagella equal Flagella different Chart 11 (=depression)

in length in length

CommonC genera examplel s:

Partlyleaf- Egg-shaped cell Leaf-shaped cell body Egg-shaped to Pseudopodia Incapabcapable ofo prp oduoduccingg

shaped cell body; with scales oval or pointed at the spherical and posteriorlyalong the ps eudopodia; with emargination

body;y; without on the cell posteriorp end;; without flattened cell gliding flagellum;; (=depression; at the flagellar

scales on the surface (visible scales on the cell surface, body; without amoeboid movements; iinsertionti alongl theth edged (“nos(“ e”))”)); cell surface; using high complex life-cycle with scales on the without emargination anteriorlydepressed laterally

threadlike magnificationifi ti ) thick cyst walls; anterior cellll surfacef ()(=depressiond i ) compressed;anterior flagellum thin

ps eudopodia flagellum typically waving often difficult to see

may be formed

Gyromitus Thaumatomastix Thaumatomonas Protaspis Bodomorpha An cyromonas Skuja 1939 Lauterborn 1899 De Saedeleer 1931 Skuja 1939 Hollande 1942 Kent 1880

ThaumatomonadsTh t d ThThaumatomonadst d ThaumatomonadsTht d CCryomonadds Gli Glissomonadsd AAncyromonadds

Common 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 sometimes 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 f lagellates 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 distinguishable by

molecular techniqu es (Arndt et al., in prep.)

Occurring bent hically; with plastids: 2 green, gold-

c oloured, sometimes olive-green, red or blue plastids

(only visible using high magnification with phase contrast);

cortex slightly metabolic (=changeable cell shape)

Presence of scales

(usua lly only visible using phase contrast)

Without scales With scales

Mode of nutrition

Heterotrophic Mixot rophic (slightly greenish or yellowish

chloroplasts 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) electron microscope

are difficult to

Variable

distingu ish 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

(shell-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 shape; flagella; lorica length

Common genera examples:

Loricavery thin; 1 Cone-shaped lorica very Lorica attached by Cone-shaped and pointed Loricalaterally compressed

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

emergent flagellum; chromatophore (mixotroph); 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

flagellum; sometimes 1 long and 1 short clearly distinct flagellum); groove; cell not forming a large

pseudopodia;7- 20μmemergent flagellum; cell forming a large lip to lipto one side; 8 - 12μm

5-28μ m one side;; 10-15μm

Cal ycomonas Poteriochromonas Stokesiella Histiona Reclinomonas

Lohmann 1908 Scherffel 1901 Lemmermann 1910 Voigt 1902 Flavin & Nerad 1993

Chrysomonads Chrysomonads Chrysomonads Jakobids Jakobids

Bicosoecid s

Common species examples:

Aqueous lorica, Bell-shaped lorica; cytostomal Bell-shaped lorica; cytostomalEllipsoidal, egg-shaped to Cylindrical lorica;

transparent; 7-10μm area usually slightly narrowed; area strongly enlarged; vase-shapedh d lorical i ; flagellum up to ca. 40μm

posterior end button-like posteri or end rounded and is been unreeled in a jerking

thickened; 12-14μm pointed slightly; 20-40μm manne r (cf. tongue 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

((LackeyL k ) BBourrellyll 195 1

Other common species examples:

B. lacustris

mighti ht alsol fform colonies, see

Chart 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; 8-20μmstar-shaped inserting; 5-20μm shaped; obligate anaerobic; 8-30μm

Collodictyon Quadricilia Multicilia lacustris Trimastix

Carter 1865 (Skuja) Vørs 1992 Cienkowsky 1881 Kent 1880

Collodictyonid s Protista incertae sedis Amoebozoans Preaxostyla

Num ber 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 Protista incertae sedis Spironemids Spironemids

2 distinct, posterior flagellaPresence of posterior flagella Without posterior flagella

CelCell l shape; flagella/cell length length Cell shape; number of flagella; cell length

Irregular posterior cell projection; 6 Ova l, spindle-shaped, cigar-shapedhd 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

DiplomonaDi l dds DiDiplomona l dds Diplomonads DiDiplomonads l d

Chart 6.

850 A. Jeuck and H. Arndt

Colonial

PPresence off collarll ; number of flagella; presence of lorica (housing); presence of

stalk

Without collar; 1 visible flagellum Without collar; 2 flagella; With collar; 1 flagellum; Without collar;2flagella (second (secondsedtoattachtothe lorica); without lorica; without stalk with or without stalk flagellum hardtosee); without lorica; with or without stalk with stalk FfForm of the colon ly; Form of the colony Form of the colony Form of the colony

with or without stalk

Colonies star-shaped: Colonies shaped Colonies first Colonies branched or Colonies branched or Colonies

6-30 cells attached byy like a tree, br anched shaped 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- sponge -like, to a stalk, some attached colonies disc-shaped to colonies are

without stalk ( free- storeyed);storeyed with long older colonies by intercellularbridgesto sphericalortubular colourless,

swimming) stalk ( attached) often llob batet eaceachh other,her otheotherr cellcellss finger-shaped to linear; older ones

radi ally, spspongongee-like or flagellum 2-3 times ofteoftenn

tu belike arranged longer than cell bodies brownish

see CCharthart 9: Colonial

choanoflagellates

BiBicosoeca llacustrit is BicosoBi eca petiolatai l SpongomonasS PhPhalansteriul i m

James-Clark 1867 (Stein) Bourrelly 1951 S tein 1878 Cienkowski 1870

Bicosoecids Bicosoec ids Spongomonadsp g Amoebozoans

WithWithout t CCommon speciesi examplel s:

stalk seseee also Phalansterium

Chart 5 digitatum

S tein 1878

Number of cells per colony

Spongomonas uvella

S tein 1878

Bicosoeca ‘socialis‘=colony

form of Bicosoeca lacustris

Co mmon species examples:

Com mon species example:

Cluster of up to 8 Number of cells often

cells or single < 60, spherical or

cells;ll iirregularlyl l hemispherical

branched system colonies; umbel-like

of rigid stalks with a branched stalk

Cells also free-

swimmin g; if

Siderodendron Anthophysa withith redd stigmati

Pringsheim 1946 Bory 1822 (eyespot(eyespot) A. steini Senn

Chrysomonadsy Chrysomonadsy 1900

Siderodendron manganiferumg Anthophysap y vegetang s

Pringsheim 1946 (Müller) Stein 1878

Single cel ls are difficult to distinguish from Spumella – like flagellates

Chart 7.

Guide to Free-living Heterotrophic Freshwater Flagellates 851

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

Cho anoflageg llates ((Crasppedida))

PresencPresenceeof of theca

((=membranous layyer,, enclosingg the cell body)y) Without theca With theca

Number of collars,collars, presence of stalk

One collar Two colla rs One collar;; stalked

MonosigaM i KKentt 18801880 DiDipl losiga i Fl1892Frenzel 1892 CdCodosiga i

Jamess-ClarClarkk1868 1868

Co mmono ssppeeccies examplese a p es: Monosiga angustata

Kent 1880 Monosiga ovata

Kent 1880

D iplosiga socialis

ColonColonyy formform of Codosiga:

FrenzelF l 18189292

Monosiga fusiformis see next Chart

KentK 1880

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

Form of ththee theca

Cup-p 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

Type single cell of the

species

species: colonyl formingf i

examplp e: Stelexomonas

dichotoma (see

Chart 9)

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

(Schilhillerl ) StStarmach 1968 JJames-ClClarkk 18186688 SSchillhiller 19531953 FottF 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,,

example for Salpingoeca (after Dayel et al. 2011)

Desmarella – like colony Sphaeroeca – like colony

CColonial choanofflagellates (C(Craspedida))

Form of colony

Unbranc hed colonies Branched colonies Slow swimmer Fast swimmer Solitary cells Colonial cells

Salpingoeca – like

thecate cell

SittinSittingg separatelyseparately or in Usually 4-4 15 cells sittingsitting togethetherr ManyM stalkedt lk d celllls radidiallyll 66-60 celcellsls maymay be

groupupss at tthehe ttiipp of thethe stalk, like a pearpearll necklace;necklace; singlesingle cells aarrangedrr anged in a ggelatinouselatinous heheldld 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

JameJ s-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

CoC mmon speciesi

example:

SiSingle l cellll

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

Codosiga botrytis those are ca pable of producing pseudopodia at the

(Ehrenberg)(Eh b ) KentK t 1880 posterior i endd.

Com mon species example:

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

long and tthinhin individuualal stalks to stalk, surrounded by a

a common stalk;; bra nched like dichotomous tube of

an umbel or bifurcated organici matter

CCodosiga d i Stelexomonas dichotoma

James-ClarkClar k 18681868 Lackey 1942

Accor ding to latest molecular investigations,

Codosiga umbellata

this morphotype is present in several cryptic

Stein 1878 speciesi whichhi h currentlytl mighi htt nott yett beb distinguished

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, vacuoles or

and freshwater environments, this guide may also extrusomes.

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

ellate groups in brackish water systems.

samples and analysis by epiﬂuorescence microscopy. The fol-

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

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

Methods Porter and Feig 1980; Sherr et al. 1989), buffered formalde-

hyde (e.g. Børsheim and Bratbak 1987; Sherr and Sherr 1983)

The guide is based on an analysis using the classical living- and 0.5% acidic Lugol’ solution (10 g I2, 20 g KI, 10 g sodium

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

Arndt and Mathes 1991; Gasol 1993; Massana and Güde et al. 1989).

◦

1991). This method is reliable to detect morphological and The ﬁxed samples need to be kept at 4 C in the dark until

behavioural characteristics of even very small HF regarding staining. Fluorescent dyes (e.g. DAPI (= 4 6-diamidino-2-

their main taxonomic groups – in contrast to hidden charac- phenylindole, Sigma Lot 47H4107, Porter and Feig 1980) are

teristics within samples treated by fixatives. Pelagic flagellates suitable for staining. After staining the sample should be filtered

␮

(undiluted samples) and benthic ﬂagellates (diluted by a fac- onto a 0.2- m membrane ﬁlter using vacuum not exceeding

tor of 5- >20 with ﬁltered water) can be studied in droplets of 150 mm Hg. For an even distribution of the particles, another

Guide to Free-living Heterotrophic Freshwater Flagellates 855

Eugleg nids

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

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

Changeability of the cell shape

Metabolic (=ch( angeable cell shape) Relatively rigid

Form/move ment of flagellum/cellg Form/movement of flagellum/cellg body;y;

body; cell shape/length cell shape/length

Common genera examples: Common genera examples:

Well developed Relatively thick flagellum Relatively thick flagellum, Flagellumstiffed in front Flagellum most active

flagellum, most active beatingbea ting in whiplawhiplashsh fashion; 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; (ro tating) or metabolically shaped, laterally or tip; egg-shaped, ‘flickering’ funnel-

cigar-, cone-shaped glid ing; spindle-shaped to ddorsoventrt allyll flattttenedd; spindle-shaped oval to shaped frontal region,

to spherical (often cyli ndrical; 16 - 80μm 14-100μm triangular; rigid cells and often striated;

deforming) often dorsoventrally flattened; 25 - 60μm

flattened; ; 20 -100μmμ 6-50μm

Peranema Astasia Menoidium Peta lomonas Urceolus

Dujardin 18411841 Dujardin 1841 Perty 1852 Stein 1859 Mereschkowsky 1879

Confusion with

Notosolenus possible:p

obser ve carefully if

there is a second

Paramylon (P)

P ffl lagellumll (when( h theth cell turns) P

ConfusionConfusion with

Heteronema possible:

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

iis a secondd flagellumfl ll c ell shape; cell length; presence of storage granule (paramylon)

(when the cell turns)turns)

Some com mon genera examples:

With ingestion apparatus (often hard to With ingestion apparatus With ingestion apapparatparatusus With ingestioningest ion apapparatparatus

see);see); swimming anterior flagellum weaker (small)(small);; swimming anterior (often hard to see); swimmingswimming (well developedeveloped d and

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

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; slightlyli htl mettabolicb li (=ch( hangeable bl ((=chhangeablble cellll shah pe);) dorsod ventrt allyll fl attttened;d rigid;i id celllls ddorsoventrally ll

cell shape)shape) or rigrigid;id; 14-100μm 2525-100μm 10-10 30μm flattened;flattened; rigid;rigid; 10 -30μm30μm

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

em. SkSkujaj 19391939

Ingestion

ConfusionCon fusion with Con Confusiofusionn with apparatust

Per anema possible Petalomonas P

if the second possible if the flagellum is second flagellum is overlooked overlooked

Second flagellg um

Chart 12.

856 A. Jeuck and H. Arndt Apusomonad s

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

Anterior flagellum liesinaproboscis (=trunk-like protrusion of the Anterior trailing flagellum protrudes from the proboscis (=trunk-like

cell); recurrent flagellum often hard to see; cells are metabolic protrusion of the cell); recurrent flagellum lies directed posteriorlypy under (=changeable 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 Alexeieff 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

Flagella length; presence of dark refracting particles (=ejectisom( es) (partlyhardto see); presence of bright

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

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

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

anterior 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; wit h 3 - 6 striations; clearly granulum; without striations; circular section; striations; circular section; swimming and

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

substrate; 5 -12μm

Goniomonas Cryptomonas Kathablepharis

(Fresen ius) Stein 1878 Ehrenberg 1831 emend. Hoef-Emden & Melkonian 2003 Skuja 1939

Synonym : Cyathomonas Fromentel 1874 Synonym: Chilomonas Ehrenberg 1831

Cryp tomonads Cryptomonads Kathablepharids

Comm on species examples: Common species example: Common species example: Movement:

Similar species fast, rotating in

Refracting

occur in brackish Manyfood a waving

ejectisomes water: vacuoles manner, shorter e.g. flflagellum ll beats b t

G. amphinema around the the Larsen & Cryptomonas paramaecium anterior end;

Patterson 1990: Ehrenberg 1832 emend. onlyyp pelagically g y

Hoef-Emden & Melkonian occu ring.

2003

Kathab lepharis Goniomonas truncata ovalis (Fresenius) Stein 1878 Skuja 1948

Chart 14.

Guide to Free-living Heterotrophic Freshwater Flagellates 857

Dinoflagellates

Cell shape

Egg-shaped,, ellipsoidal to spherical Other cell shape

Position of the equatorial groove (cingulum) OthOther positioniti off theth Anterior position of

cingulum the cingulum

CellC ll shah pe; fform off grooves; celllll length th

Co mmon ggenera examples:p

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

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

longitudinal groove ( sulcus)l ) posteriort i end,d ththe twot endsd projections or wings; cingulumi l almostl t edged s of the grooves are

partially spirals to cingulum, ofthe cingulum meet; horizontally divides the body into two clearly visible due to ridges;

the two ends of the cingulum morphologmorphologyy chchangangees due to halfs of different length; 20-100μm

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

50μm

Gyrodinium Gymnodinium Peridiniopsis Peridinium

Kofoid & Swezyy 1921 Stein 1878 Lemmermann 1904 Ehrenbergg 1832

With With

Without

WitWithouthout chlhloroplastl ts chl hloroplastl ts

c hloroplastp s, chloroplasts, but may but may

contain containt i

c oloul 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 dodorsoventrallso e t a y flatteneatte edd;; 88-50μ50μm the cingulum withowithoutut torsion;torsion; 6-6 50μ m

Without With

chloroplastp s c hloroplasts Amphidinium

Katodinium Claparède & Lachmann

Fott 195719 57 1858/61

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

Co mmon genera examples:

With longer or shorter horn-like Lenss- shaped to roroundund, almost Elongateelliptical or lunate,

ppr ojectionj s (ante( rior end with CommCommonon symmetrical;y ; 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 theth cellll bodyb d ;

(cingulum)cingulum ) lies horizohorizontallyntally, 1616-40μm With

divides the cell body into two chloroplasts

hal fs of nearlyy eqqual length;g Without 40-450μ m chloroplashl l ts,t but may

With containcontain

chloroplasts c oloured

CCeratiumti Diplopsal is Cystodinium

Cera tium hirundinella food

Schrank 1793 Bergh 1881 Klebs 1912

(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.

membrane (0.6 ␮m pore size) may be placed underneath the

◦ Appendix A. Supplementary data

ﬁlter. The ﬁlters are generally stored deep-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 http://dx.doi.org/10.1016/j.protis.2013.08.003.

parts of raw samples). This should aid in morphotype iden-

tiﬁcation of bacterivorous forms and to check whether the

determined morphotypes are life-cycle forms (swarmer, colony

etc.) of other types, as it is often the case with e.g. choanoﬂag-

ellates where extrinsic factors can induce the formation of References

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