The D1-D2 region of the large subunit ribosomal DNA as barcode for ciliates

The D1-D2 region of the large subunit ribosomal DNA asbarcode for ciliates

T. STOECK,* E. PRZYBOS† and M. DUNTHORN*

*Department of Ecology, University of Kaiserslautern, 67663 Kaiserslautern, Germany, †Institute of Systematics and Evolution of

Animals, Polish Academy of Sciences, 31-016 Krak�ow, Poland

Abstract

Ciliates are a major evolutionary lineage within the alveolates, which are distributed in nearly all habitats on our

planet and are an essential component for ecosystem function, processes and stability. Accurate identification of

these unicellular eukaryotes through, for example, microscopy or mating type reactions is reserved to few specialists.

To satisfy the demand for a DNA barcode for ciliates, which meets the standard criteria for DNA barcodes defined

by the Consortium for the Barcode of Life (CBOL), we here evaluated the D1-D2 region of the ribosomal DNA large

subunit (LSU-rDNA). Primer universality for the phylum Ciliophora was tested in silico with available database

sequences as well as in the laboratory with 73 ciliate species, which represented nine of 12 ciliate classes. Primers

tested in this study were successful for all tested classes. To test the ability of the D1-D2 region to resolve conspecific

and congeneric sequence divergence, 63 Paramecium strains were sampled from 24 mating species. The average con-

specific D1-D2 variation was 0.18%, whereas congeneric sequence divergence averaged 4.83%. In pairwise genetic

distance analyses, we identified a D1-D2 sequence divergence of <0.6% as an ideal threshold to discriminate Parame-

cium species. Using this definition, only 3.8% of all conspecific and 3.9% of all congeneric sequence comparisons had

the potential of false assignments. Neighbour-joining analyses inferred monophyly for all taxa but for two Parame-

cium octaurelia strains. Here, we present a protocol for easy DNA amplification of single cells and voucher deposi-

tion. In conclusion, the presented data pinpoint the D1-D2 region as an excellent candidate for an official CBOL

barcode for ciliated protists.

Keywords: Ciliophora, D1-D2 region, DNA barcode, LSU-rDNA, single-cell PCR, voucher deposition

Received 24 June 2013; revision received 20 October 2013; accepted 21 October 2013

Introduction

Despite an increased importance of species identification

for much biological research (Hebert et al. 2003a), there

is a worldwide shortage of essential taxonomic training

and information (Schander & Willassen 2005; Guerra-

Garc�ıa et al. 2008). To countervail this taxonomic impedi-

ment, biological identifications through DNA barcodes

have been introduced (Hebert et al. 2003a,b). DNA bar-

coding uses short, standardized gene regions as internal

species tags to provide rapid identifications (Hebert &

Gregory 2005). By facilitating taxonomy, DNA-barcoding

approach has found numerous initiatives that have

mainly targeted multicellular organisms; for example,

fish (April et al. 2011), insects (Burns et al. 2005), birds

(Hebert et al. 2004), mammals (Borisenko et al. 2008),

plants (Kress et al. 2005) and fungi (Seifert et al. 2007).

Microbial eukaryotes (protists) have thus far largely

been ignored in large collaborative barcoding initiatives

and projects, although they are more diverse than multi-

cellular eukaryotes (Patterson 1999; Pawlowski et al.

2012), distributed in nearly all habitats (Epstein & L�opez-

Garc�ıa 2008) and are an essential component for ecosys-

tem processes and stability (Corliss 2002). One major

reason for this lack of barcodes in nonphotosynthetic

microbial eukaryotes is that they are not monophyletic;

rather, they are distributed in all super groups in the

eukaryotic tree of life (Simpson & Roger 2004; Koonin

2010; Adl et al. 2012; Pawlowski et al. 2012). Their geno-

mic diversities are too divergent to find a single locus

that serves as a barcode for all of them. This lack of

protists in DNA-barcoding initiatives becomes evident

from the bibliography of International Barcode of Life

Project (http://ibol.org/barcoding-bibliography), which

lists 1236 peer-reviewed DNA-barcoding publications

between the years 2003 and 2012, only a negligible

proportion (<2%) of which target protists.Correspondence: T. Stoeck, Fax: +49-631-2052496;

E-mail: [email protected]

© 2013 John Wiley & Sons Ltd

Molecular Ecology Resources (2014) 14, 458–468 doi: 10.1111/1755-0998.12195

Given this, under the empowerment of the Interna-

tional Nucleotide Sequence Database Collaboration

(Cochrane et al. 2011), the Consortium for the Barcode of

life (CBOL) established the Protist Working Group

(ProWG) with the ultimate objective to establish univer-

sal criteria for barcode-based species identification in

protists (Pawlowski et al. 2012). ProWG proposed a two-

step pipeline: first, use the hyper-variable V4 region of

the small subunit ribosomal DNA (SSU-rDNA) locus to

assign an isolate to a major taxonomic group (e.g. Bacil-

lariophyceae, Cercozoa, Ciliophora, and Dinoflagellata);

second, apply a group-specific barcode, which is

acknowledged and accepted by the scientific community

working with this taxonomic group (Pawlowski et al.

2012). Reliable and promising barcode regions for some

protist groups are already established; for example, helix

37 of the SSU-rDNA for foraminifera (Pawlowski &

Lecroq 2010). Most protist taxa, though, are still awaiting

an adequate DNA barcode.

Here, we suggest a barcode marker that shows high

potential for identifying ciliate species. Ciliates are a

large protist clade that is recognized by the presence of

micronuclei and macronuclei within each cell (Lynn

2008). Many species can be identified morphologically

(Lynn 2008), which can serve as the basis for DNA bar-

coding, even though cryptic species are also known (Son-

neborn 1937, 1957; Nanney 1999; Simon et al. 2008). Up

to 40 000 estimated ciliates species (Nanney 2004; Foiss-

ner et al. 2008) are central players in the microbial loop

in most ecosystems (Azam et al. 1983; Finlay & Fenchel

1996; Corliss 2002).

The most frequently used barcode for ciliates is COI

(Lynn & Str€uder-Kypke 2006; Chantangsi et al. 2007;

Gentekaki & Lynn 2009; Str€uder-Kypke & Lynn 2010;

Kher et al. 2011; Greczek-Stachura et al. 2012). Other

genes have been analysed for their potential as DNA bar-

codes for ciliates: for example, nuclear ribosomal internal

transcribed spacer regions (Barth et al. 2006; Gentekaki &

Lynn 2009; Greczek-Stachura et al. 2012); nuclear histone

H4 (Greczek-Stachura et al. 2012), and the mitochondrial

cytochrome b (Lynn & Str€uder-Kypke 2006; Barth et al.

2008; Przybo�s et al. 2010)—none of which, however, met

CBOL’s approval criteria for non-COI barcodes (http://

barcoding.si.edu/pdf/dwg_data_standards-final.pdf).

These criteria include (i) ease of DNA extraction and

sequencing; (ii) primer and gene universality; (iii) the

presence of a barcode gap; and (iv) voucher deposition

with type species, or tissue sample.

Recently, Santoferrara et al. (2013) suggested the

D1-D2 region of the large subunit ribosomal DNA (LSU-

rDNA) as barcode for tintinnids, an important and

abundant clade of planktonic ciliates (Dolan et al. 2013).

Using the tintinnids as a test clade, the D1-D2 region of

LSU-rDNA was able to better distinguish among species

than SSU-rDNA, and the hyper-variable V4 and V9

regions of SSU-rDNA (Santoferrara et al. 2013). In this

study, we further analyse the D1-D2 region of the LSU-

rDNA to evaluate whether this gene region meets the cri-

teria for a general ciliate barcode marker as defined by

the Consortium for the Barcode of Life.

Materials and methods

In silico analyses to test PCR-primer specificities

As an initial test of the ability to amplify the D1-D2

region from a broad range of taxa, LSU-rDNA sequences

of available ciliates were downloaded from GeneBank’s

nucleotide (nr) database using the search operator ‘[Cil-

iophora(Organism)] AND (LSU OR 28S) NOT (ITS1 OR

internal OR protein OR 16S OR 18S OR mitochondrial

OR 5.8S)’ and aligned with Muscle (Edgar 2004) as

implemented in SEAVIEW v. 4 (Gouy et al. 2010) against

the PCR primers used in this study, specific for the

D1-D2 region of the LSU-rDNA for all eukaryotes

[forward primer: 5′- AGCGGAGGAAAAGAAACTA-3′;

and reverse primer 5′- ACGATCGATTTGCACGTCAG-

3′) (Sonnenberg et al. 2007)]. The number of sequences in

the alignment was 65, representing the ciliate classes

Colpodea, Heterotrichea, Litostomatea, Nassophorea,

Oligohymenophorea, Plagiopylea, Prostomatea and

Spirotrichea (Table S1, Supporting information). Only

four classes (Armophorea, Cariacotrichea, Karyorelictea

and Phyllopharyngea) were not represented in this data

set. The total length for this alignment was 4276 positions

(longest sequence = Ichthyophthirius multifiliis, GenBank

Accession no. EU185635.1 with 2677 bp).

Laboratory experiments to test primer specificities

We tested primer universality in PCRs with DNA from

49 different ciliates from seven of 12 major ciliate clades

(sensu Adl et al. 2012) (Table 1). DNAs originated from

taxa collected and provided by Wilhelm Foissner

(University Salzburg, Austria) and Bettina Sonntag

(University Innsbruck, Austria) and previously

sequenced for the SSU-rDNA locus at the Department of

Ecology, University of Kaiserslautern, for phylogenetic

analyses. The PCR mix included dNTPs (10 lmol each,

200 lM final, Axon, Germany), 100 lmol/lL of a Fw1

and Rev2 primers from Sonnenberg et al. (2007) (each

0.5 lM final), 0.5 lL HotStar Taq (5 U/lL, 2.5 U final,

Qiagen, Germany) and 5 lL of 109 Coralbuffer (19 final,

Qiagen). The reaction mix was filled with sterile water to

a final volume of 50 lL. The PCR protocol comprised an

initial denaturation at 95 °C for 5 min, followed by 30

identical amplification cycles of denaturation at 95 °C for

1 min, annealing at 64 °C for 1 min, and extension at

© 2013 John Wiley & Sons Ltd

D1 -D2 REGION AS CIL IATE BARCODE 459

72 °C for 1 min, followed by a final extension at 72 °Cfor 10 min. PCR product was purified with the MiniElute

Kit (Qiagen) and cloned into a vector using the TA-Cloning

Kit (Invitrogen, Carlsbad, CA). To check for any intra-

polymorphisms, seventeen randomly chosen samples

(Table 1) of the resulting PCR products were purified

Table 1 Ciliates from seven different classes that were tested in PCR for the D1-D2 region of the LSU rDNA (Sonnenberg et al. 2007)

Taxon name Class Collected and identified GenBank Accession no.

Bryometopus sp. Colpodea WF

Bursaria sp. 1 Colpodea WF

Bursaria sp. 2 Colpodea WF

Colopda henneguyi Colpodea WF

Colpoda maupasi Colpodea WF

Colpoda minima Colpodea WF

Isiella palustris Colpodea WF

Maryna umbrellata Colpodea WF

Pseudomaryna sp. Colpodea WF

Woodruffides metabolicus Colpodea WF

Spirostomum ambiguum Heterotrichea WF KF287645

Spirostomum teres Heterotrichea BS KF287659

Stentor coeruleus Heterotrichea BS KF287658

Stentor muelleri Heterotrichea BS KF287653

Fuscheria terricola Litostomatea WF

Monodinium sp. Litostomatea WF

Monodinium sp. Litostomatea BS

Pelagodileptus trachelioides Litostomatea BS

Spathidium cf. fraterculum Litostomatea WF

Trachelophyllum sp. Litostomatea BS KF287655

Bromeliophrya sp. MD2012 Oligohymenophorea WF KF287646

Cinetochilum margaritaceum Oligohymenophorea BS KF287654

Dexiotricha sp. Oligohymenophorea WF

Dexiotricha tranquilla Oligohymenophorea BS

Epistylis sp. Oligohymenophorea WF

Glaucomides sp. 2 Oligohymenophorea WF KF287660

Glaucomides sp. 1 Oligohymenophorea WF KF287647

Lambornella sp. 1 Oligohymenophorea WF KF287644

Lambornella sp. 2 Oligohymenophorea WF

Ophryoglena sp.1 Oligohymenophorea BS

Ophryoglena sp.2 Oligohymenophorea BS KF287656

Paramecium tetraurelia Oligohymenophorea KL

Pseudocohnilembus sp. Oligohymenophorea WF KF287649

Telotrochidium sp. Oligohymenophorea WF KF287648

Tetrahymenid ciliate Oligohymenophorea WF KF287651

Urocentrum turbo Oligohymenophorea BS KF287650

Vorticella convallaria Oligohymenophorea WF

Vorticella convallaria Oligohymenophorea BS

Vorticella sp. Oligohymenophorea WF

Tokophrya infusionum Phyllopharyngea WF

Coleps hirtus cf. viridis Prostomatea BS KF287657

Gastrostyla sp. Spirotrichea WF

Gonostomum sp. Spirotrichea WF KF287652

Orthoamphisiella stramenicola Spirotrichea WF

Oxytricha c.f Spirotrichea WF

Oxytricha ottowi Spirotrichea WF

Oxytricha ottowi Spirotrichea WF

Oxytricha sp. Spirotrichea WF

Sterkiella cf. caviola Spirotrichea WF

For details, see Materials and methods section. All taxa produced PCR bands of the expected size. Sixteen randomly chosen PCR prod-

ucts were chosen for sequencing, all of which resulted in the correct D1-D2-sequence. Accession nos are provided in the last column. BS,

Bettina Sonntag, University of Innsbruck; KL, Stoeck lab, University of Kaiserslautern; WF, Wilhelm Foissner, University of Salzburg

© 2013 John Wiley & Sons Ltd

460 T . STOECK, E . PRZYBOS and M. DUNTHORN

with the MiniElute Kit (Qiagen), cloned into a vector

using the TA-Cloning Kit (Invitrogen, Carlsbad, CA) and

sequenced using vector primers with the Big Dye termi-

nator chemistry (Applied Biosystems, Foster City, CA)

on an ABI 3730 automated sequencer.

Testing the barcoding gap in Paramecium

Strain selection. To test the ability of the D1-D2 region to

resolve conspecific and congeneric sequence divergence,

63 Paramecium strains were sampled from 24 mating spe-

cies (Table 2). These strains are in the permanent culture

collection of the Polish Academy of Sciences, Institute of

Systematics and Evolution of Animals; they are available

from the authors upon request. Cells were grown at

room temperature in Volvic water, amended with a

wheat grain and Klebsiella minuta as a food source.

To use a protocol applicable to environmental

samples without prior cultivation, a single-cell PCR was

conducted for D1-D2 PCR amplification. An individual

cell was picked from a culture, then washed in sterile

Volvic water. The cell, in a volume of 5 lL of sterile

washing water, was then transferred into a PCR tube

containing the reaction mixture as described previously.

PCR protocol, purification of PCR products, cloning and

sequencing followed the protocol as described earlier.

We note that cloning is not an essential step in this proto-

col. Alternatively, PCR products from single cells can be

successfully sequenced directly when using the PCR-

primers as sequencing primers. However, to allow for

long-term material storage (plasmids with inserts) in our

laboratory, we preferred the cloning step. All obtained

sequences went through rigorous standard quality

assessments, PHRED and PHRAP analysis using the

program CODONCODE ALIGNER v. 1.2.4 (CodonCode

Corporation, Dedham, MA). GenBank Accession nos are

provided in Table 2.

Sequence analyses. Pairwise genetic distances of the

resulting Paramecium sequences were calculated with

PairAlign as implemented in JAguc (Nebel et al. 2011).

Pairwise distances were written in a triangular distance

matrix and used to calculate intra- (conspecific) and

interspecific (congeneric) variation in the D1-D2 frag-

ment in the strains used in this study. For the neighbour-

joining (NJ) analyses, the D1-D2 LSU-rDNA sequences

of the Paramecium strains (Table 2) were aligned in SEA-

VIEW v. 4 (Gouy et al. 2010) using Muscle (Edgar 2004).

The alignment was manually refined in MacClade

(Maddison & Maddison 1992) and start- and end-

trimmed to the same position in all sequences. The final

alignment included 799 positions and is available from

the authors upon request. An NJ tree was constructed

under the K2P evolutionary model as recommended by

Table 2 Paramecium (sibling) species, strains, and origins used

in this study.

Species

Origin of strains and

[strain number]

GenBank

Accession

no.

P. primaurelia Sevilla, Andalusia, Spain

[3/1]

KF287661

Valmanara, Italy [4/1] KF287662

Near Rejkjavik, Iceland [5/

1]

KF287663

Piekary near Krak�ow,

Poland [6/1]

KF287664

Nałezcz�ow (Lublin region),

Poland [7/1]

KF287665

Hanoi, Vietnam [17/1] KF287666

Onoda, Japan [18/1] KF287667

P. biaurelia Tasmania Island, Australia

[2/2]

KF287668

Yamaguchi, Japan [3/2] KF28769

Marlishausen, Germany

[6/2]

KF287670

Velke Heraltice, Czech

Republic [7/2]

KF287671

Krak�ow, Poland [13/2] KF287672_Zywiec Beskids, Poland

[14/2]

KF287673

Astrahan Nature Reserve,

Russia [16/2]

KF287674

P. triaurelia San Rafael, Spain [3/3] KF287675

Krak�ow-Opatkowice,

Poland [6/3]

KF287676

Natural Reserve Complex

Volga-Ahtuba, Russia [9/

3]

KF287677

P. tetraurelia Sydney, Australia [1/4] KF287678

Botanical Garden,

Melbourne, Australia [2/

4]

KF287679

Tabgha, Israel [6/4] KF287680

Paris, France [8/4] KF287681

Skalnate Pleso, Tatras,

Slovakia [9/4]

KF287682

Botanical Garden, Krak�ow,

Poland [10/4]

KF287683

P. pentaurelia Pennsylvania (87), USA

[1/5]

KF287684

Vaciamadrid, Rivas, Spain

[2/5]

KF287685

Valmarana, Italy [3/5] KF287686

Balaton Lake, Hungary [4/

5]

KF287687

Astrahan Nature Reserve

(AZ6-24), Russia [6/5]

KF287688

Altai Foreland, Russia [7/

5]

KF287689

P. sexaurelia Puerto Rico (159), Spain

[1/6]

KF287690

KF287691

© 2013 John Wiley & Sons Ltd

D1 -D2 REGION AS CIL IATE BARCODE 461

CBOL (http://barcoding.si.edu/pdf/dwg_data_standards-

final.pdf).

Results and discussion

To demonstrate that the D1-D2 region of the LSU-rDNA

locus is an appropriate barcode for ciliates, we will walk

through the main criteria for a successful DNA-barcod-

ing as specified by CBOL (http://barcoding.si.edu/pdf/

dwg_data_standards-final.pdf).

Ease of DNA extraction and sequencing of D1-D2

Ciliates range in their size between c. 10 lm (some scuti-

cociliates) up to c. 4 mm (some Spirostomum species)

(Lynn 2008). They are characterized by ‘germline’

micronuclei and ‘somatic’ macronuclei, the latter of

which possesses tens to thousands of copies (Jahn &

Klobutcher 2002; Gong et al. 2013). Such high genome

copy numbers in ciliates make DNA extractions

un-needed as they are ideally suited for single-cell PCR’s

(Lynn & Pinheiro 2009) (see Fig. S1, Supporting informa-

tion). Specific genes, including potential barcoding

genes, are accordingly highly replicated in the macronu-

clei of ciliates and provide sufficient template for PCR

amplification. This is specifically helpful when it comes

to ciliates that are difficult to culture or directly isolated

from an environmental sample for species identification.

This ease of single-cell PCR amplification in ciliates

has been taken advantage of in a number of studies (e.g.

Gong et al. 2013). Yet, we note that without doubt, sin-

gle-cell PCR’s are easier to perform on larger ciliate cells,

and genes from very small species may be more difficult

to amplify in single-cell reactions. As a solution to this

problem, we suggest whole-genome amplification

(WGA), which performs well with minute DNA concen-

trations prior to targeted PCR. The length of suggested

barcode here is about 840 bp. This corresponds approxi-

mately to the length of the COI gene fragment length

used as potential barcodes in ciliates (Gentekaki & Lynn

2009) and is about 190 bp more than the COI region used

for vertebrate and insect barcoding (Hebert et al. 2004;

Wiemers & Fiedler 2007). Such a fragment length is still

possible to sequence with one single Sanger read, a strat-

egy that complies with the recommended CBOL protocol

for sequence analyses (http://www.barcodeoflife.org/

content/about/what-dna- barcoding). In case of direct

sequencing of PCR products without prior plasmid

cloning, such a read length may be critical. Therefore, we

recommend cloning of PCR products for this specific

DNA-barcoding protocol. In case of direct PCR-product

sequencing, it would be beneficial to assess whether a

shorter fragment of the D1-D2 region, sequenced from

the 5′ or 3′ end would suffice for species discrimination.

Table 2 (Continued)

Species

Origin of strains and

[strain number]

GenBank

Accession

no.

Phuket Island, Thailand

[2/6]

Yamaguchi, Japan [3/6] KF287692

Joannina, Greece [5/6] KF287693

Seville, Spain [6/6] KF287694

Stuttgart, Germany [8/6] KF287695

Astrahan Nature Reserve,

Russia [9/6]

KF287696

P. septaurelia Natural Reserve Volga-

Ahtuba (AZ6-23), Russia

[2/7]

KF287697

P. octaurelia Florida (138), USA [1/8] KF287698

Ein Effek, Israel [2/8] KF287699

P. novaurelia Lafiloliere (534), France

[1/9]

KF287700

P. decaurelia Florida (223), USA

[1/10]

KF287701

P. undecaurelia Texas, (219), USA [2/11] KF287702

P. dodecaurelia Elba Island, Italy [4/12] KF287703

Jordan’s Park, Krak�ow,

Poland [7/12]

KF287704

P. tredecaurelia Paris (209), France [1/13] KF287705

Cuernavaca (321), Mexico

[2/13]

KF287706

Kyryat Motzkin, Israel [3/

13]

KF287707

P. quadecaurelia Namibia Vindhoek, Africa

[2/14]

KF287708

P. sonneborni Texas, USA, ATCC

30995

KF287709

P. bursaria Syngen 3, Bejing, China

[1/3(b)]

KF287710

Syngen 4, Oklahoma,

Ardmoore, USA [1/4(b)]

KF287711

Syngen 5, St.Petersburg,

Russia [1/5(b)]

KF287712

P. calkinsi Vladivostok, Maritime

Territory, Russia

[2/cal]

KF287713

P. caudatum Titicaca, Peru [4/c] KF287714

P. jenningsi Bangalore, India [2/j] KF287715

Okinawa, Japan [3/j] KF287716

P. multimicronucleatum Rome, Italy [1/m] KF287717

Cheboksary, Russia

[2/m]

KF287718

Baton Rouge, Louisiana,

USA [4/m]

KF287719

P. nephridiatum Pisa, Italy [P.n.] KF287720

P. polycarium Khabarovsk, Russia [P.p.] KF287721

P. putrinum Khanka Lake, Russia

[P.put.]

KF287722

P. woodruffi Slavyanka, Maritime

Territory, Russia [P.w.]

KF287723

© 2013 John Wiley & Sons Ltd

462 T . STOECK, E . PRZYBOS and M. DUNTHORN

Primer and gene universality of D1-D2

Nuclear protein-coding genes emerged as too conserved

for intraspecific analyses in ciliates (Gentekaki & Lynn

2009). They are also subject to extensive paralogy and

rapid rates of evolution (Israel et al. 2002; Katz et al.

2004; Aury et al. 2006; Dunthorn & Katz 2008). While

mitochondrial genes, especially COI, have been shown to

be effective as a barcode, this region can hardly be ampli-

fied in the large and ecologically important clade Spiro-

trichea (Str€uder-Kypke & Lynn 2010). Another problem

with COI is that many ciliates are found in anoxic habi-

tats (Stoeck et al. 2007; Lynn 2008; Orsi et al. 2012), and

thus lack functional mitochondria and the full set of

mitochondrial genes, such as COI. While it may be a

good genetic marker to identify populations, species and

cryptic species in some ciliates, COI is not an effective

barcode for all ciliates because mitochondria are missing

is many ecologically important taxa (Lynn 2008). Given

CBOL’s criteria for DNA barcodes, COI will have to be

rejected as a general ciliate barcode marker.

The D1-D2 region of the LSU-rDNA, on the other

hand, is found in all ciliates, and thus is potentially a bet-

ter general ciliate barcode. Here, for in silico analyses of

primer specificities, we have retrieved sequences from

only 65 different species from the GenBank database

(Table S1, Supporting information). Of these, 43 species

included the original D1-forward region and 34 species

included the region of the D2-reverse primer (Sonnen-

berg et al. 2007). Alignments showed a coverage of 35%

for the original D1-forward primer and of 18% for the

original D2 reverse primer. However, degenerating the

original D1-forward primer at two positions to 5′-AG-

CGGAGGARAAGAAAHTA-3′ results in a 96% coverage

for the forward primer. Only the two species Entodinium

sp. (Accession no.: Z49857.1) and Epidinium sp.

(Z49914.1) remain with one mismatch to the degenerate

forward primer. Both species are trichostomatide ciliates

belonging to the Litostomatea. Similarly, degeneration of

the reverse primer at one position to 5′- ACGADCGA

TTTGCACGTCAG-3′ increases the target coverage of

this primer to 85%. The five sequences that still show

mismatches to the degenerate primer are from Apodioph-

rys ovalis (JF694045.1), Epiphyllum shenzhenense (JF975392.

1), Loxophyllum jini (JF975393.1), Phialina salinarum

(JF975395.1), and Loxophyllum sp. (JF975388.1). The latter

four species are all haptorid ciliates belonging to the

class Litostomatea, while A. ovalis is a Spirotrichea.

We also found in laboratory experiments that the pri-

mer pairs were able to produce PCR products of the

expected size from all 49 test strains using the original

D1-D2 primers. Of these, all seventeen PCR products

randomly chosen for sequencing were identified as the

correct D1-D2 regions (Table 1). Thus, we were able to

recover the D1-D2 region of LSU-rDNA from nine of

twelve classes of Ciliophora (Cariacotrichea, Armopho-

rea, and Karyorelictea excluded) in silico and in labora-

tory experiments, indicating good primer universality

within the ciliates.

Presence of a barcode gap in D1-D2

Ideally for a barcode, there should be a ‘barcode gap’,

for which genetic variation within a biological species

is lower than divergence among biological and cryptic

species (Hebert et al. 2003a). For the identification of

species in birds, Hebert et al. (2004) proposed that

within genera (congeneric) sequence divergences

should be one order of magnitude greater than within

species (conspecific) sequence divergence. Typically, in

a variety of animal phyla, the intraspecific COI diver-

gence is less than 2%, while the average interspecific

COI sequence divergence is commonly more than 8%

(Hebert et al. 2003b).

For ciliates, a barcode gap for the D1-D2 region of

LSU-rDNA was demonstrated for tintinnid ciliates (San-

toferrara et al. 2013). Here, we show that this gap also

occurs in closely related, and oftentimes cryptic, Parame-

cium species. The average conspecific D1-D2 variation is

0.18%, whereas congeneric sequence divergence aver-

ages 4.83%. In detail, 96.2% of the conspecific pairwise

sequence comparisons (n conspecific pairwise sequence

comparisons = 105) have a sequence divergence in the

D1-D2 region <0.6% (Fig. 1). Only four pairwise

sequence comparisons show a divergence that is >0.5%.

One pair of Paramecium bursaria (syngen three from

China and syngen five from Russia) exhibits 3.8%

sequence divergence in the D1-D2 region. A recent study

demonstrated that P. bursaria seems to be a species com-

plex consisting of different species (Greczek-Stachura

et al. 2012). However, unlike the recognized species of

the P. aurelia complex, the P. bursaria complex is only

recognized and officially described as one species. We

therefore consider the different syngens of the P. bursaria

complex as one species, even though the D1-D2 fragment

of the LSU-rDNA confirms that P. bursaria is a cryptic

species complex. Three pairwise sequence comparisons

within P. multimicronucleatum show a D1-D2 sequence

divergence >0.6% (Fig. 1). Therefore, it is reasonable to

assume that also P. multimicronucelatum is a species

complex rather than one defined species. This is also

evidenced by COI and ITS gene analyses, in which

P. multimicronucleatum strains show high intraspecific

variations (Barth et al. 2006). Our data provide further

support that concerted efforts of ciliate taxonomists

should be to officially erect species complex status for

P. bursaria and P. multimicronucleatum and define indi-

vidual species according to the Code of the International

© 2013 John Wiley & Sons Ltd

D1 -D2 REGION AS CIL IATE BARCODE 463

Commission of Zoological Nomenclature (http://iczn.

org), as carried out for P. aurelia species.

In case of tintinnid ciliates, which are morphologically

identified by a lorica, not present in other ciliates, Santof-

errara et al. (2013) used 1% sequence divergence (pair-

wise p-distance) in the D1-D2 region of the LSU-rDNA to

discriminate the maximum number of species. At this

cut-off, only 14% of all species could be falsely assigned.

We here identify a D1-D2 sequence divergence of <0.6%as an ideal threshold to discriminate Paramecium species.

Using this definition, only 3.8% of all conspecific

sequence comparisons have the potential of false assign-

ments. Likewise, in the congeneric D1-D2 sequence

analyses, 3.9% of all pairwise comparisons (n

total = 1845) show a divergence of <0.6% and could be

falsely assigned. Even though the vast majority of all con-

generic sequence pairs (n congeneric Paramecium

sequence comparisons in this analysis = 1561) diverge

for >1% in their D1-D2 LSU-rDNA region, the risk of

false assignments would increase from 3.9% to 15% for

pairwise congeneric sequence comparisons when using a

sequence divergence cut-off of 1%.

The risk of falsely positive or negative assignment is

low compared with other cases without a true barcoding

gap. Just to name a few examples, in Lepidoptera of the

genus Agrodiaetus, overlaps in the range of intra- and

interspecific COI sequence divergence is 18% (Wiemers

& Fiedler 2007). In an analysis of more than 400 Diptera

species, the success rate of species identification using

COI was even lower than 70% (Meier et al. 2006), and

COI barcoding in marine gastropods show a 16% chance

of false assignments (Meyer & Paulay 2005). Also, Cnida-

ria show a much higher risk of false assignments (Hebert

et al. 2003b) and several orders of insects show substan-

tial overlap in conspecific and congeneric COI sequence

divergence resulting in 45% false assignments (Cognato

2006). Hardly, any such comparative data are available

for protists. An exception are diatoms, that are subject

of relatively intense barcoding efforts (Moniz &

Kaczmarska 2009, 2010; Hamsher et al. 2011; Zimmer-

mann et al. 2011; Saunders & McDevit 2012). Several

different barcode markers tested in distinct genera of dia-

toms show a much higher risk of false assignments due

to a larger overlap between conspecific and congeneric

sequence divergences (Moniz & Kaczmarska 2009). More

promising in diatoms seems the V4 region of the SSU-

rDNA, which identified almost all of the 123 limnetic

diatom species analysed by Zimmermann et al. (2011).

In cases where a barcoding gap does not exist, evolu-

tionary models are suggested as alternative strategies for

species diagnosis (Austerlitz et al. 2009; Lou & Golding

2010). The phylogenetic analyses conducted with the Par-

amecium sequences confirm the suitability of the D1-D2

region as barcode marker for Paramecium (Fig. 2). With

one exception (Paramecium octaurelia), this evolutionary

model reliably resolves cryptic species and morphospe-

cies. Using these molecular data, 61 species were inferred

to be monophyletic. The error rate of 3.1% is thus in the

same order of magnitude as for genetic distances. By

contrast, in a corresponding tree profile approach, which

relies on taxon sampling and the monophyly of species

rather than a barcoding gap, at least 16% of Agrodiaetus

specimens were misidentified (Wiemers & Fiedler 2007).

Our results for Paramecium and the D1-D2 region of LSU-

rDNA corroborate with the requirements of CBOL for

the performance of a genetic barcode marker.

Voucher depositions

One major advantage of ciliates compared with other

microbial eukaryotes is their relative ease of enrichment,

isolation and cultivation, although some species most

0

10

20

30

40

50

60

70

80

90

100%

Spe

cim

en p

airs

Category (% sequence divergence)

ConspecificCongeneric

Fig. 1 D1-D2 LSU rDNA sequence diver-

gence (%) between conspecific and conge-

neric pairs of Paramecium sequences (see

Table 1). The average conspecific D1-D2

variation is 0.18%, whereas congeneric

sequence divergence averages 4.83%. The

vast majority (n = 1561) of all congeneric

sequence pairs diverge for >1% in their

D1-D2 LSU region. While most (93.3%)

conspecific sequence pairs diverge not

more than 0.5%. Defining a barcode gap

at 0.6% sequence divergence in the D1-D2

region, c. 3.5% of all data have the poten-

tial of false assignments. The total number

of congeneric sequence divergence com-

parisons runs to 1845 and for conspecific

comparisons this number is 105.

© 2013 John Wiley & Sons Ltd

464 T . STOECK, E . PRZYBOS and M. DUNTHORN

likely remain recalcitrant to the laboratory. Diagnostic

microscopy slides can thus be prepared from barcoded

and identified cultures for type species deposition, using

standard silver staining procedures (e.g., Foissner 1991).

Another advantage of ciliates is their relatively large cell

size, which makes them comparably easy to spot in envi-

ronmental samples. This is helpful for ciliates that escape

cultivation efforts.

DNA barcoding of individual ciliates, though, results

in destroyed cells. Diagnostic images are therefore

suggested as being acceptable as depository material

(Pawlowski et al. 2012). To achieve this aim, we take

advantage of a method of Auinger et al. (2008), which

combines Lugol’s iodine staining of whole samples or

individual cells, followed by microscopy analyses and

imaging with subsequent single-cell PCR of the imaged

cell (Fig. S1, Supporting information). This way, a spe-

cific morphotype can be linked to a specific barcoded

genotype. We note that this strategy does not allow

naming new species, but it is still useful for the identifi-

cation of known morphotypes and deposited DNA bar-

codes and also helps to discover novel and undescribed

diversity.

Outlook

The primer set used in this study successfully amplified

the D1-D2 region of the LSU-rDNA from 73 ciliate spe-

cies (Paramecium strains included) originating from seven

distinct ciliate classes. Also in silico analyses of available

ciliate LSU-rDNA sequences in public databases show

that a substantial proportion of these sequences include

the complementary annealing sites for the D1-D1-primer

pair tested in this study. However, considering the high

diversity of these protists, further primer tests with more

taxa will be necessary and when indicated, the modifica-

tion of these primers or the design of novel primers

targeting this D1-D2 region. Likewise, further efforts will

Fig. 2 Neighbour-joining Kimura 2-parameter evolutionary model tree of the Paramecium sequences obtained and analysed in this

study. In cases with more than one sequence per species, the concept of a monophyletic species as prerequisite for barcoding is met.

One exception is the two strains of Paramecium octaurelia, which do not fall into other monophyletic clades, but which do not branch

together in monophyly. For details on tree construction, see Materials and methods section.

© 2013 John Wiley & Sons Ltd

D1 -D2 REGION AS CIL IATE BARCODE 465

be to evaluate whether conspecific and congeneric

distances in the D1-D2 region in other ciliate genera are

in the same order of magnitude as reported here for

Paramecium. Finally, it will be a major task to fill the cili-

ate D1-D2 barcode database with data. The number of

globally described free-living ciliate species is about 4500

(Foissner 2008) included in c. 1500 genera (Aescht 2001).

Estimates are that the number of free-living ciliate

species may be even as high as 40 000 (Foissner et al.

2008). This sharply contrasts with the number of D1-D2

rDNA sequences of described ciliate species deposited in

public databases (n = 65 in Genbank database on April

30, 2013). Even though this latter task will be a major

basic research endeavour, which requires the contribu-

tion of numerous scientists in this field, it will lay the

cornerstone for numerous applications and pave the way

for ciliate species diagnosis in a variety of fields with

high relevance for science, economy and politics.

Acknowledgements

Funding for this study came from the Deutsche Fors-

chungsgemeinschaft (DFG grant STO414/3-1) to T.S. and

(DFG grant DU1319/1-1) to M.D. The authors thank

Tobias Siemensmeyer, Franziska G€odecke and Isabell

Trautmann for help with labwork. Furthermore, we

express our gratitude to Bettina Sonntag (University of

Innsbruck, Austria) and Wilhelm Foissner (University

of Salzburg, Austria) for the identification and providing

of ciliate species for gene analyses. We also thank Alexey

Potekhin (St. Petersburg State University, Russia) for

strains of Paramecium bursaria, P. calkinsi, P. polycarium,

P. putrinum, and P. woodruffi from Core Facilities Centre

“Culture Collection of Microorganisms” used in the

studies.

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T.S. conceived and designed the study, E.P. collected,

identified and provided Paramecium strains, M.D. and

T.S. contributed to laboratory work, T.S. analysed data,

T.S. and M.D. wrote manuscript, T.S. and M.D. super-

vised the work.

Data Accessibility

Sequences were deposited in the GenBank database

under Accession nos KF287645- KF287652 (see Table 1)

and KF287661- KF287723 (see Table 2). Phylogenetic tree

is available from TreeBase under http://purl.org/

phylo/treebase/phylows/study/TB2:S14701.

Supporting Information

Additional Supporting Information may be found in the online

version of this article:

© 2013 John Wiley & Sons Ltd

D1 -D2 REGION AS CIL IATE BARCODE 467

Fig. S1 Flow chart for barcoding ciliates. Single cells are isolated

from environmental samples, enrichments or pure cultures,

stained with 10% LUGOL solution and photographed under the

light microscope. The same individual target cells are destained

in sodium thiosulphate for protocol see (Auinger et al. 2008) and

subjected to PCR with primers targeting the D1-D2 region of

the LSU rDNA. Gene fragments are Sanger-sequenced and

deposited as voucher along with other information (Pawlowski

et al. 2012).

Table S1 Ciliates including the D1-primer region or the D2-pri-

mer region or the complete D1-D2 fragment of the LSU rDNA

and accession numbers available in GenBank (April 30, 2013)

and used for in silico primer analysis.

© 2013 John Wiley & Sons Ltd

468 T . STOECK, E . PRZYBOS and M. DUNTHORN