Genetic Diversity in the Paramecium aurelia Species Complex

Genetic Diversity in the Paramecium aurelia Species Complex

Francesco Catania,* Francxois Wurmser,�� Alexey A. Potekhin,§ Ewa Przybos,k andMichael Lynch*

*Department of Biology, Indiana University; �Laboratory Evolution, Genomes and Speciation; CNRS-UPR9034, Gif sur YvetteCedex, France; �Universite Paris-Sud 11, 91405 Orsay Cedex, France; §Laboratory of Protozoan Karyology, Faculty of Biology andSoil Science, St Petersburg State University; Oranienbaumskoye sh., 2, Stary Peterhof, 198504 Saint Petersburg, Russia; andkDepartment of Experimental Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Krakow, Poland

Current understanding of the population genetics of free-living unicellular eukaryotes is limited, and the amount ofgenetic variability in these organisms is still a matter of debate. We characterized—reproductively andgenetically—worldwide samples of multiple Paramecium species belonging to a cryptic species complex, Parameciumaurelia, whose species have been shown to be reproductively isolated. We found that levels of genetic diversity both inthe nucleus and in the mitochondrion are substantial within groups of reproductively compatible P. aurelia strains butdrop considerably when strains are partitioned according to their phylogenetic groupings. Our study reveals the existenceof discrepancies between the mating behavior of a number of P. aurelia strains and their multilocus genetic profile,a controversial finding that has major consequences for both the current methods of species assignment and the speciesproblem in the P. aurelia complex.

Introduction

Protists are among the most abundant and diverse eu-karyotic groups (Patterson 1999), yet current knowledge ofthese organisms is surprisingly sparse, compared with thatfor animals, plants, and fungi. Two aspects of microbial eu-karyotes that still remain controversial are the level of spe-cies diversity and their geographic distribution (Finlay andFenchel 1999; Foissner 1999). One school of thought main-tains that protist species are relatively few, cosmopolitan,and ubiquitous (Fenchel et al. 1997); another supports es-sentially the opposite view, that is, protist species are nu-merous and tend to be endemic and isolated (Foissner2006). The currently limited understanding of the protistworld (Weisse 2008) makes it difficult at this time to sup-port one hypothesis or the other, in part due to the paucity ofpopulation-genetic studies that could shed light on theglobal (effective) population sizes of free-living species.

In a species, the genetic effective population size (Ne)(Wright 1931)—which modulates the efficiency of naturalselection via its effects on random genetic drift (Ohta1992)—reflects the number of breeding individuals as wellas aspects of the breeding system, population structure, anddegree of linkage, and 4Nel (where l is the neutral muta-tion rate) is the expected neutral genetic variation (averagenucleotide heterozygosity per site) of a diploid populationat mutation–drift equilibrium. As estimates of l are avail-able for a number of species (Lynch 2006a), measures ofneutral genetic variation (e.g., diversity at synonymoussites in protein-coding genes) allow Ne to be inferred indi-rectly from estimates of variation at silent sites in naturalpopulations.

Although large-scale polymorphism studies of protistpathogens (e.g., Grigg et al. 2001; Mu et al. 2002) have pro-vided measures of neutral genetic variation (Lynch andConery 2003; Lynch 2006b), the neutral genetic variationof pathogenic organisms is expected to be biased downward

and not directly applicable to free-living taxa. On the otherhand, a number of small-scale polymorphism surveys ofprotein-coding loci in free-living microbes, freshwater cil-iates in particular, have produced conflicting estimates ofintraspecific variation (Gerber et al. 2002; Barth et al.2006; Katz et al. 2006; Snoke et al. 2006), thus impedingwider conclusions.

Not only can polymorphism surveys based on singleor a few genes produce estimates of 4Nel that are poten-tially misleading, but poorly defined species boundariesalso make the estimation of intraspecific variation problem-atic. The latter issue may well represent a confounding fac-tor in the computation of genetic diversity for microbialorganisms (Daubin and Moran 2004), as the process of‘‘species’’ identification is a difficult task, given the frequentexistence of cryptic species (Waugh 2007), and may requirea combination of approaches that include molecular, mor-phological, and ecological studies (Weisse 2008).

In this sense, the free-living Paramecium is a very suit-able system, as the ‘‘species problem’’ in this ciliate group,and in P. aurelia in particular, has been intensely investi-gated. Paramecium aurelia is a complex that contains 15species (Sonneborn 1975; Aufderheide et al. 1983). Despitebeing morphologically identical, these species are geneti-cally isolated and, to quote Sonneborn (1975), ‘‘the distin-guishing characters of each of the [then 14] species includeits mating reactions, breeding relations, and mode of inher-itance of mating type.’’ Specifically, each of the P. aureliaspecies contains two mating types—E (for ‘‘even’’) and O(for ‘‘odd’’)—that determine intraspecific sexual reproduc-tion by conjugation (Sonneborn 1937). When two clonesthat have either the same mating type or belong to differentspecies are mixed, no conjugation occurs. Although weakcross-reactions between distinct mating types of differentspecies may be observed (e.g., between Paramecium tet-raurelia and Paramecium octaurelia or between Parame-cium primaurelia and Paramecium pentaurelia) (Beale andPreer 2008), these reactions result in the death of the con-jugating cells or in the sterility of their F1s. The reproduc-tive cohesion of P. aurelia species (Sonneborn 1975), theavailable macronuclear genome sequence of P. tetraurelia(Aury et al. 2006), and a variety of worldwide samples col-lected during both the past century and in very recent years,

Key words: Paramecium, genetic diversity, effective populationsize, speciation, cryptic species, mating group switching.

E-mail: [email protected].

Mol. Biol. Evol. 26(2):421–431. 2009doi:10.1093/molbev/msn266Advance Access publication November 20, 2008

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make this species complex an appealing candidate for apopulation-genetic study.

In the attempt to shed light on extant controversial re-sults (Katz et al. 2006; Snoke et al. 2006) and to expand ourknowledge on the levels of genetic diversity in free-livingunicellular eukaryotes, we performed a polymorphism sur-vey of multiple nuclear and mitochondrial loci in a large setof P. aurelia strains. Our analysis aims to overcome poten-tial limitations of previous studies on free-living unicellulareukaryotes, such as limited sample sizes and both restrictednumber of genes and sampled locations. Along with a num-ber of insights to the population genetics of multiple speciesof the P. aurelia complex, we found a surprising incongru-ence between mating behavior and strains’ genetic profiles,which complicates definitive conclusions on intraspecificlevels of genetic variability.

Material and MethodsParamecium Strains

We examined a total of 80 Paramecium strains, 79members of the P. aurelia species complex (Sonneborn1975; Aufderheide et al. 1983) (table 1), and 1 Parameciummultimicronucleatum strain. A total of 26 strains werekindly provided by J. Cohen, H. Schmidt, M. Simon,T. Berendonk, and K. Aufderheide, whereas the remaining54 strains are maintained in the Collection of Parameciumof the Laboratory of Protozoan Karyology, St PetersburgState University. All strains are fully homozygous, follow-ing multiple rounds of autogamy.

Culturing and Identification of Paramecium Strains

Paramecia were cultured in a medium made of driedlettuce (or Yeast extract, Cerophyl, Na2HPO4, and Stigmas-terol) and distilled water inoculated with Enterobacteraerogenes and identified according to the methods ofSonneborn (1970). Clones matured for conjugation weremated with the reactive complementary mating types ofstandard strains of all species of the P. aurelia complex.

In intrastrain and interstrain crosses, the F1 generationwas obtained by conjugation and F2 by autogamy (using themethod of daily isolation lines). The occurrence of the de-sired stage of autogamy (specimens at the stage of two mac-ronuclear anlagen) was examined on preparations stainedwith aceto-carmine. Survival of clones in both generationswas estimated as percentages. According to Chen (1956),clones can be considered as surviving after passing sixto seven fissions during 72 h after separation of partnersof conjugation or postautogamous caryonides. The methodsare described in detail in Przybos (1975).

DNA Extraction and Gene Sequencing

Genomic DNA extraction was performed by incubat-ing Paramecium cells for 20 min at 90 �C with 5% Chelex100 Resin. We surveyed genetic variability at 10 nuclearand 5 mitochondrial protein-coding loci (table 2). In addi-tion, we sequenced three rDNA fragments: a portion of the

nuclear small rDNA subunit (SSU), the two internal tran-scribed spacers (ITS1 and ITS2), and a mitochondrial frag-ment encompassing the 5.8S and large rDNA subunit(LSU) (table 2).

We used the P. tetraurelia macronuclear (http://para-mecium.cgm.cnrs-gif.fr/) (Aury et al. 2006) and mitochon-drial (Pritchard et al. 1990) genome sequences and thefreely available online software ‘‘Primer3’’ (Rozen andSkaletsky 2000) to design DNA oligo pairs to coding re-gions for polymerase chain reaction (PCR) amplifications(table 2 and supplementary table 3 in SupplementaryMaterial online). More than a single pair of oligos was oftenrequired to amplify a locus across most or all strains sur-veyed. The nuclear loci studied were random single-copygenes in the P. tetraurelia macronuclear genome. We am-plified the gene regions using the following PCR condi-tions: 3 min at 94 �C, 40 cycles of 40 s at 94 �C, 40 sat 55 �C, and 1 min at 72 �C. Amplification products weresubsequently checked on 1% agarose gels, and reactionsgiving unique products were sequenced in both directions.We verified the quality of the sequenced gene fragmentsboth automatically, using the software Codon CodeAligner, and visually. We used ClustalW (Thompsonet al. 1994), as implemented in the software Bioedit (Hall1999), to align the consensus sequences.

Data Analysis

Sequences were exported to the software MEGA 4.0(Nei and Kumar 2000) for data analysis. For each protein-coding locus, we measured intraspecies and interspecies di-versity at both silent sites and replacement sites, followingthe Kumar method (Nei and Kumar 2000). Diversity valueswere multiplied by n/(n � 1), where n is the appropriatenumber of strains for each species, to correct for small sam-ple size, and the average values were weighted by the num-ber of base pairs sequenced for each locus. The MaximumComposite Likelihood model implemented in MEGA 4.0and bootstrap statistics (1,000 replicates) were used to buildNeighbor-Joining (NJ) trees using all the sites of the regionsexamined. We also measured diversity in single or concat-enated intronic regions (the latter when genes containedmore than one intron) contained in each of the surveyedloci, after removing the conserved GT–AG ending sites.The package GENECONV (Sawyer 1999) was employedto estimate both the number and the location of possiblerecombinant alleles.

All the sequences generated in this study were submit-ted to GenBank, accession numbers FJ002921–FJ004151.

Results and DiscussionHigh Levels of Silent Genetic Variation withinReproductively Compatible P. aurelia Strains

Levels of intraspecific diversity at nuclear synony-mous sites (ps) in the P. aurelia species complex average0.1493 (SEM 5 0.0382) (table 3 and Supplementary Ma-terial online). Diversity at silent sites is ;13-fold higherthan at nonsynonymous sites (pa 5 0.0117 [SEM 50.0031]) but is comparable with that calculated for intronic

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sites (pi 5 0.0944 [SEM 5 0.0246]). No intron presence orabsence polymorphisms were detected across species, andlevels of diversity in coding and intronic regions show sig-nificant positive correlations when estimates for single lociare examined (pa vs. pi, Spearman’s rho 5 0.643, P, 0.01;ps vs. pi, Spearman’s rho 5 0.540, P , 0.01). In the mi-tochondria, loci also showed a substantial degree of varia-tion (ps 5 0.3257 [SEM 5 0.0674] and pa 5 0.0173[SEM 5 0.0057]), with silent sites having on average;2.2 times the nucleotide diversity of nuclear genes (table4 and Supplementary Material online). Levels of net inter-specific divergence (i.e., in excess of within-species diver-gence) at silent sites average 0.3307 (SEM 5 0.0209) forthe nuclear loci (supplementary table 1 in SupplementaryMaterial online) and 0.5214 (SEM 5 0.0184) for mitochon-drial loci (supplementary table 2 in Supplementary Materialonline). At amino acid replacement sites, divergence dropsto 0.0239 (SEM 5 0.0012) and 0.0175 (SEM 5 0.0175) fornuclear and mitochondrial loci, respectively. The ratio ofmitochondrial to nuclear divergence at (neutrally evolving)silent sites reflects the ratio of the mutation rates in the twocellular compartments and averages 4.0223 (SEM 52.6470) when all species are examined but decreases to1.2504 (SEM 5 0.2451) after the removal of Parameciumsexaurelia, a species previously reported to show unusuallydivergent genotypes across different geographic locationsand where F2 generations of interstrain crosses showlow rates of survival (Stoeck et al. 1998; Przybos, Rautian,et al. 2007).

Table 1List of Paramecium Strains/Species Surveyed

Strain Species Origin

16Paramecium

primaurelia Woodstock, MD, United States168 Japan33 Unknown60 United States61 Unknown90 Pennsylvania, United StatesAZ15-8 Astrakhan Nature Reserve, RussiaAZ9-3 Astrakhan Nature Reserve, RussiaIr4-1 The Irtysh river, Omsk, RussiaKK2-7 Kaliningrad region, RussiaTB10 Rejkjavik, IcelandV7-6 Volgograd region, Russia

223-7The Volga river, Tver’region, Russia

112-1 Vologda Region, Russia227 Unknown

1038sParamecium

biaurelia Unknown

114Bloomington, IN, United

States (uncertain)AB7-16 Boston, United StatesAZ25-2 Astrakhan Nature Reserve, RussiaF2 UnknownGA62-1 Altai Mountains, RussiaKr133-2 Krasnoyarsk, RussiaP2 UnknownPK2 Krakow, PolandRR2-1 Saint-Petersburg region, RussiaTB15 Teneriffa, Canary Islands, SpainUM2 UnknownV1-4 Volgograd region, RussiaW7 Unknown

152alphaParamecium

triaurelia New Haven, United States

EP_2/3Natural reserve Complex

Volga-Ahtuba, Russiakr128 Krasnoyarsk, RussiaKr149-1 Krasnoyarsk, RussiaTB17 GreeceTB18 Santuhalm near Deva, RomaniaV10-7 Volgograd region, Russia

172Paramecium

tetraurelia Peru298s Unknown32 Unknown51s Indiana, United Statesd4-2 —nr7-1 Novorossiysk, RussiaPF15 Paris, FranceS Sydney, AustraliaST Tatry, SlovakiaBGD19 Unknown

AZ6-24Paramecium

pentaurelia Astrakhan Nature Reserve, RussiaGA1-9 Altai Mountains, RussiaNr1-10 Novorossiysk, RussiaRA81-8 Altai Forelands, RussiaV2-7 Volgograd region, Russia

159Paramecium

sexaurelia UnknownAZ8-4 Astrakhan Nature Reserve, RussiaCB16-2 Beijing, China

AZ5-2Paramecium

septaurelia Astrakhan Nature Reserve, RussiaAZ8-3 Astrakhan Nature Reserve, RussiaGFg-1 Freiburg, GermanyV5-13 Volgograd region, Russia

Table 1Continued

Strain Species Origin

138Paramecium

octaurelia Florida, United States565s UnknownIEE Ein Efek, IsraelK8 UnknownK9 Unknown

AB8-22Paramecium

novaurelia Boston, United StatesIz16 Izmail, UkraineV9-6 Volgograd region, RussiaVL4-8 Vladimir, Russia

223Paramecium

decaurelia Florida, United States93-21 Yaroslavl region, RussiaGA1-12 Altai Mountains, RussiaJN Nara, Japan

219Paramecium

undecaurelia United States

246Paramecium

dodecaurelia Mississippi, United States256-2 Yaroslavl region, RussiaUV1-3 Vinnitsa, Ukraine

209Paramecium

tredecaurelia Paris, France

328Paramecium

quadecaurelia AustraliaAN1 Namibia

Parameciumsonneborni Texas, United States

Parameciummultimicronucleatum Krasnoyarsk, Russia

The Global Effective Population Size of Paramecium 423



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Low Levels of Genetic Variation within MonophyleticTaxa

The analysis of the gene genealogies across all nuclearand mitochondrial loci revealed that 13 ‘‘discordant’’ strainsconsistently group with species that are different from thatto which they were originally assigned by mating tests(table 5 and supplementary fig. 1 in Supplementary Mate-rial online). We verified this unexpected finding by furtherstudying the macronuclear SSU and ITSs and the mitochon-drial 5.8 and LSU sequences (Supplementary Material on-line) and reperforming mating experiments on this set ofsuspect strains. We conclude that although these strainscontain a multilocus genetic profile that is highly similarto that of a given P. aurelia species, they preferentially matewith strains belonging to a different species within the spe-cies complex. As it is legitimate to question whether ornot these discordant strains ought to be included in the cal-culation of intraspecific genetic variability, we also calcu-lated levels of polymorphism after their exclusion. Wefound that this leads to a considerable decrease inwithin-species genetic variability, with an average ps of

Table 2Paramecium Genes Surveyed and Primers Used for PCRAmplification

Nuclear Loci Molecular Function PCR Primers (5#–3#)

GSPATG00000151001

DNA mismatch repair

system Msh2

gagagacatctaaactgtgcgtttt

tggcattttgatccatcttc

GSPATG00000223001

Calcium binding

protein

ttccctgaccgaataggattt

ggccataagcatccaagatt

GSPATG00000363001 tRNA methyltransferase

gcagcagctgttggtcataa

tccagtgtgtccataagcttaattt

GSPATG00000454001

GTP-binding protein

RAB2 homolog

tgaacatgatgcaactattggag

gtggatttgcaggtttgcta

GSPATG00000546001

GrpE protein

homolog

tcccttgatgaatctcacga

tgttcctggttctttttcagg

GSPATG00027725001

3-Oxoadipate

enol-lactonase

ttggcaggatggtctttagg

tggcatatgaccaacacctg

GSPATG00028095001

Serine

carboxypeptidase

ttacatcattagtgtttgtcaggtg

cctgcataactctccccaaa

GSPATG00028110001 Hypothetical protein

agttggcgataatggaggac

aaccatcagctccatcaacc

GSPATG00022332001

Ubiquitin-like 1

activating

enzyme E1B

tggcattggataatgcagaa

gtagaggcaatggcgtgaat

GSPATG00020758001

Hypothetical

protein

aagccacctccagattttca

tttcccagatcgaacctttg

Mitochondrial loci

NADH 3

tgggtagtatgacattgcttttc

ggccagcgagttctggac

ORF189-2 & yegR

gccctctcagagctcaacat

acaccgggtggacgtaga

COX I

ccaaaattggggttacgatg

attgcgtttttgaggacgac

COX II

gcatagaagtagtcatcagcaacc

ttttgtgaggttgtatttccaca

rDNA loci

SSUa (nuclear)

gcaagtctggtgccagcagcc

cttccgtcaattcctttaag

ITS–5.8S rDNA–ITSa

(nuclear)

tcctccgcttattgatatgc

ggaagtaaaagtcgtaacaagg

5.8S-LSU

(mitochondrial)

ttggttgagggcgtaaatct

ctaccccgcacaaaagaaaa

a Primer sequences match NS3, NS4, ITS4, and ITS5 in White et al. (1990).

Table

3IntraspecificGenetic

Diversities

atNonsilentSites

(pa),SilentSites

(ps),andIntronic

Sites

(pi)withCorrespondingStandard

Errors

(SE),Estim

atedforAllStrainsacross

10

NuclearLoci

Sp

ecie

s

All

Str

ain

sO

nly

No

nd

isco

rdan

tS

trai

ns

Av

erag

eN

o.

of

Str

ain

sp

a(S

E)

ps

(SE

)p a

/ps

pi

(SE

)A

ver

age

No.

of

Str

ain

sp a

(SE

)p s

(SE

)p

a/p

sp i

(SE

)

Param

ecium

primaurelia

12

0.0

01

1(0

.00

03

)0

.024

8(0

.00

58

)0

.044

40

.024

9(0

.01

42)

11

0.0

01

0(0

.00

03)

0.0

22

8(0

.00

55)

0.0

43

90

.02

40

(0.0

14

4)

Param

ecium

biau

relia

10

0.0

13

1(0

.00

47

)0

.168

3(0

.06

95

)0

.077

80

.130

2(0

.10

10)

10

0.0

00

4(0

.00

03)

0.0

03

8(0

.00

15)

0.1

05

30

.00

75

(0.0

09

5)

Param

ecium

triaurelia

70

.00

29

(0.0

00

7)

0.0

34

4(0

.00

69

)0

.084

30

.016

7(0

.00

83)

60

.000

3(0

.00

03)

0.0

06

6(0

.00

25)

0.0

45

50

.00

10

(0.0

01

3)

Param

ecium

tetraurelia

10

0.0

07

6(0

.00

13

)0

.090

8(0

.01

16

)0

.083

70

.044

6(0

.01

30)

90

.000

7(0

.00

04)

0.0

07

9(0

.00

28)

0.0

88

60

.00

40

(0.0

03

4)

Param

ecium

pentau

relia

40

.01

08

(0.0

05

6)

0.1

20

8(0

.05

55

)0

.089

10

.041

6(0

.03

51)

40

.000

9(0

.00

12)

0.0

01

7(0

.00

21)

0.5

29

40

.00

36

(0.0

04

6)

Param

ecium

sexaurelia

30

.00

03

(0.0

00

6)

0.0

03

8(0

.00

79

)0

.078

90

.005

8(0

.01

66)

30

.000

3(0

.00

06)

0.0

03

8(0

.00

79)

0.0

78

90

.00

57

(0.0

16

3)

Param

ecium

septau

relia

40

.01

97

(0.0

04

0)

0.2

74

8(0

.04

70

)0

.071

70

.151

4(0

.05

05)

30

.000

4(0

.00

03)

0.0

03

4(0

.00

24)

0.1

17

60

.00

53

(0.0

06

0)

Param

ecium

octaurelia

50

.00

57

(0.0

01

4)

0.0

74

3(0

.00

81

)0

.076

70

.049

8(0

.02

11)

40

.000

3(0

.00

02)

0.0

03

6(0

.00

16)

0.0

83

30

.00

85

(0.0

06

9)

Param

ecium

novaurelia

30

.01

66

(0.0

03

7)

0.2

56

1(0

.03

36

)0

.064

80

.201

2(0

.11

28)

20

0.0

09

1(0

.01

30)

0.0

00

00

Param

ecium

decaurelia

30

.02

94

(0.0

09

4)

0.2

94

0(0

.07

11

)0

.100

00

.245

1(0

.11

41)

20

.002

3(0

.00

20)

0.0

10

4(0

.00

68)

0.2

21

20

.01

09

(0.0

18

4)

Param

ecium

dodecaurealia

30

.03

10

(0.0

06

4)

0.4

15

4(0

.07

40

)0

.074

60

.197

1(0

.08

49)

20

.000

8(0

.00

09)

0.0

07

8(0

.00

61)

0.1

02

60

.01

09

(0.0

15

8)

Param

ecium

quad

ecau

relia

20

.00

19

(0.0

04

0)

0.0

34

5(0

.01

15

)0

.055

10

.024

1(0

.08

11)

20

.001

9(0

.00

40)

0.0

34

5(0

.01

15)

0.0

55

10

.02

45

(0.0

82

3)

1 Av

erag

e0

.01

17

(0.0

03

1)

0.1

49

3(0

.03

82

)0

.075

1(0

.00

43)

0.0

94

4(0

.02

46)

0.0

00

8(0

.00

02)

0.0

09

6(0

.00

28)

0.1

22

6(0

.04

01

)0

.00

88

(0.0

02

3)

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0.0096 (SEM 5 0.0028) and 0.0460 (SEM 5 0.0177) in thenucleus and in the mitochondrion, respectively (tables 3 and 4and Supplementary Material online). The net interspecific av-erage divergence at silent sites increases to 0.4034 (SEM 50.0198) and to 0.6717 (SEM 5 0.0203) at nuclear and mito-chondrial loci, respectively (supplementary tables 1 and 2 inSupplementary Materialonline), but it remains similar atnon-synonymous sites (0.0293 [SEM 5 0.0012] and 0.0269[SEM 5 0.0016]). In addition, levels of diversity at intronicsites and at amino acid replacement sites are no longer signif-icantly correlated (pa vs. pi, Spearman’s rho 5 0.032,P 5 0.772; ps vs. pi, Spearman’s rho 5 0.292, P , 0.01).The ratio of mitochondrial-to-nuclear divergence at silentsites averages 3.8049 (SEM 5 2.5900) and decreases to1.1597 (SEM 5 0.4212) after the removal of P. sexaurelia.

Discordant and Phylogenetically Distinct P. aureliaLines

The existence of distinct and reproductively isolatedspecies (or ‘‘varieties’’ or ‘‘syngens’’ as originally reported[Sonneborn 1937, 1957]) within the P. aurelia complex hasbeen convincingly described (Sonneborn 1975) and corre-lated with molecular makers by many workers (e.g., Stoeckand Schmidt 1998). Yet, as reported above, our polymor-phism survey reveals that a number of P. aurelia species arenot monophyletic. Allele sharing between P. aurelia spe-cies has been detected by previous studies (Coleman2005; Hori et al. 2006; Snoke et al. 2006; Tarcz et al.2006; Barth et al. 2008), but little significance has typicallybeen given to this finding. The limited number of species,strains, or loci in these earlier investigations leads to a num-ber of nonmutually exclusive interpretations of the data,including 1) the occurrence of recent speciation events;2) a large Ne, whereby strains still contain segregating al-leles that predate species divergence (i.e., transspecies poly-morphisms); and 3) incomplete reproductive isolation.

Our analysis crucially shows that allele sharing be-tween strains with different mating behavior is not sporadicbut rather extends across all macronuclear and mitochon-drial loci surveyed. Also, allele sharing does not involvethe whole set of strains for a species but often concernsall nondiscordant strains for one species and only a singlestrain for a second species. Finally, distinct P. aurelia spe-cies tend to form separate clades—after the exclusion of thediscordant strains—with very short internal branches.

Consistent with a number of previous findings (e.g.,Haggard 1974), our observations seem to rule out the pos-sibility of recurrent gene flow among P. aurelia species, asthe genetic profile of the surveyed strains is never discon-tinuous, that is, we do not find evidence of introgressivehybridization or episodes of cross-species recombination.The hypothesis of transspecies polymorphisms due toa large Ne in Paramecium also seems unlikely, given themodest level of intraspecific genetic variability (5 4Nel)calculated after the removal of the discordant strains.Finally, the hypothesis of interspecific allele sharing dueto recent speciation could well explain the intimate phylo-genetic relationship between Paramecium septaurelia and-Paramecium octaurelia, whose strains tend to consistentlygroup together across all gene genealogies but hardlyT

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explains the allele sharing between multiple strains of onespecies and an isolated strain of a second species.

A more parsimonious explanation for our observationsis that the locus (loci) that is responsible for mating behav-ior in Paramecium—whose identity is still unknown—hasundergone changes in the discordant strains that switchtheir mating preference. These changes would only alterthe mating preference of these strains, which would other-wise normally react with genetically similar conspecificstrains, leaving intact the overall genetic profile. It is nota-ble that all 13 discordant strains show apparently normalviability in their matings to genetically distinct species(supplementary table 4 in Supplementary Material online).

Although not reported for any other ciliate or, to ourknowledge, eukaryotic species, the incongruence we ob-serve between mating behavior and genetic profile maynot be limited to the P. aurelia species complex, and ‘‘spe-cies switches’’ might at least extend to other ciliates withcryptic species (e.g., P. multimicronucleatum, Tetrahymenapyriformis). Under this hypothesis, it would be informativeto study the genetic profile of species other than P. aureliaand examine whether conspecific strains (following suc-cessful mating reactions) carry alleles that tend to be highlydivergent at multiple loci while similar to alleles of a relatedspecies, an analysis that is currently hampered in ciliates bythe dearth of available molecular markers and/or large-scalepopulation-genetic surveys. If the discordance betweenmating preferences and genetic profile is confirmed to becaused by an alteration of the mating locus (loci), then mat-ing locus switches could be considered a novel mechanismof speciation that, for its effects, could be comparable withother known mechanisms, such as, for example, cytoplas-mic incompatibility (Bordenstein et al. 2001).

If the above hypothesis is correct, the genetic and eco-logical effects of two distinct genetic profiles, combined inone nucleus, remains an issue to be addressed in future in-vestigations. Future studies will determine whether the F1generation obtained from such a cross is fertile (they do sur-vive autogamy) and less or more vigorous than the parentalstrains. In addition, it is currently not known if the pro-nounced interspecific sequence divergence would disfavorhomologous recombination—thus gene flow and introgres-sion—as has been observed for other organisms (Datta et al.

1997; Lukacsovich and Waldman 1999; Opperman et al.2004), and/or if the set of genes of one of the two conjugatingstrains might be invariably discarded (gynogenesis or hy-bridogenesis) (Macgregor and Uzzell 1964; Schultz 1969;Mantovani and Scali 1992; Ragghianti et al. 2007). The latterevent emerges as a possibility from earlier studies, wherea cross between two P. aurelia species, P. tetraurelia, andP. octaurelia was reported to produce survivor clones thatare not true hybrids and behave as one or the other species(the true F1 hybrids were sterile) (Levine 1953; Haggard1974). Also, unless gynogenesis or hybridogenesis occurs,these discordant species would lead to introgression (whichwe have not observed) if they generated fertile progeny.

Finally, it is worth noting that even after the exclusion ofdiscordant strains, P. aurelia species do not always formmonophyletic groups. This can be seen, for example, in thecase of P. primaurelia, where strain 227 branches off alone,makingthistaxonparaphyletic.Levelsofsequencedivergencebetween this P. primaurelia strain and all other conspecificstrains are higher, both for macronuclear and mitochondrialloci, than the divergence estimated between P. primaureliaandP.pentaurelia.Strain227alsodivergesconsiderablyfromitsotherconspecificstrainsat rRNAloci,where it tends toclus-ter with Paramecium triaurelia strains (data not shown). It istempting to conjecture that this P. primaurelia strain—of un-known geographic origin—may represent an example of in-cipient speciation in the P. aurelia complex.

Identification of Paralogous Loci

Paramecium tetraurelia has undergone at least threewhole genome duplications (WGDs), the most recent ofwhich has been suggested to coincide with the burst of spe-ciation events that led to the emergence of theP. aurelia spe-cies complex (Aury et al. 2006). An erroneous sampling ofWGD-associated paralogs in our study could have inflatedthe true genetic variation at macronuclear loci (but not atmitochondrial loci), affecting both intraspecific and interspe-cific estimates of diversity. The strain we used to design PCRprimers (d4-2, P. tetraurelia) does not contain macronuclearparalogs for the loci we have sequenced. Nevertheless, wecannot rule out the possibility that eitherP. tetraurelia strainsother than d4-2 or the remaining P. aurelia species are not

Table 5List of Discordant Strains

StrainSpecies According to

Mating TestsSpecies According to mtrRNAs and Nuclear ITS

Species According to MultipleNuclear and mt Loci

61 Paramecium primaurelia P. primaurelia/Paramecium pentaurelia P. pentaureliaPK2 Paramecium biaurelia Paramecium septaurelia/Paramecium octaurelia P. septaurelia/P. octaureliaGA62-1 P. biaurelia Paramecium triaurelia P. triaureliaTB18 P. triaurelia P. primaurelia P. primaureliaST Paramecium tetraurelia P. primaurelia P. primaureliaGA1-9 P. pentaurelia Unclear UnclearAZ8-3 P. septaurelia P. primaurelia P. primaureliaIEE P. octaurelia P. tetraurelia P. tetraureliaAB8-22 Paramecium novaurelia P. primaurelia P. primaureliaV9-6 P. novaurelia P. primaurelia P. primaureliaJN Paramecium decaurelia P. primaurelia P. primaurelia223 P. decaurelia Unclear Unclear246 Paramecium dodecaurelia P. septaurelia/P. octaurelia P. septaurelia/P. octaurelia

426 Catania et al.



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paralog-free. Also, divergent resolution of paralogous genesfollowing polyploidization and speciation (Lynch andConery 2000; Lynch and Force 2000b) might facilitate suchan erroneous sampling, but whether or not such a modelapplies to the P. aurelia complex is yet unknown.

We found two cases in which we PCR amplified paral-ogous loci and we removed these cases from our analyses. Atlocus GSPATG00022332001—the only nuclear intronlessgene we surveyed—DNA sequencing revealed the existenceof multiple double peaks in single-band PCR products ob-tained for the P. biaurelia species. We scored double peaksfor all but five of the P. biaurelia strains, and we only in-cluded these five strains in the final analyses. As single-geneduplications appear to be uncommon in Paramecium (Auryet al. 2006) and no other double-peaked DNA sequenceswere observed for other species at this locus, this additionalgene copy in P. biaurelia may be in the process of being lost

from the genome. Alternatively, this gene could be a youngduplicate that arose in P. biaurelia. GSPATG00022332001is also the only gene for which we could not obtain any PCRproduct in P. primaurelia (notably, strain 227 was the onlyone producing a band inP. primaurelia). Overall, our obser-vations suggest that GSPATG00022332001, which codesfor a protein that is involved in the ubiquitin cycle, is an evo-lutionarily labile gene in P. aurelia.

We detected the second case of paralogy after inspectingpossible inconsistencies among gene genealogies. In particu-lar, a phylogenetic tree constructed with five concatenated mi-tochondrial protein-coding loci strongly supports a closerelatedness between P. primaurelia and P. pentaurelia andthis relationship was supported by a tree constructed fromthree concatenated nuclear loci (fig. 2) and is observed forall nuclear loci but one, namely, GSPATG00028095001.At this locus, strains belonging to P. pentaurelia still cluster

FIG. 1.—NJ tree built by concatenating three mitochondrial protein-coding loci, NADH dehydrogenase subunit 3 (NADH3), cytochrome c oxidasesubunit I (COXI) and II (COXII) (1,057 bp). Only nondiscordant strains are included in the analysis. Bootstrap values equal to or higher than 75 areshown. Tetrahymena pyriformis used as outgroup.




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together (they share identical sequences) but are isolatedfrom all other species in the tree. The level of divergenceat silent sites between P. primaurelia and P. pentaurelia isas high as 80% at GSPATG00028095001 but drops to anacross-loci average of ,7% when this locus is removedfrom the analysis. We observed no PCR band at this locusforP. pentaureliawhen we used an additional PCR primer thatis external to the former pair. Although further experimentsare needed to unequivocally show that the orthologous locusto the P. tetraurelia‘s GSPATG00028095001 is not presentin P. pentaurelia, our observations are consistent with adifferential retention of paralogous loci between the twoP. aurelia species at this locus.

Molecular Phylogeny, Mating Features, and Speciationin the P. aurelia Species Complex

We obtained well-supported phylogenetic relation-ships only for the P. aurelia species that have diverged most

recently (figs. 1 and 2). In these cases, the groupingsbroadly reflect the tendency that species have to cross-reactduring mating experiments (Sonneborn 1975). In particular,P. primaurelia and P. pentaurelia as well as P. tetraureliaand P. octaurelia, which pair closely in all gene genealo-gies, are known to cross-react (the latter pair more stronglythan the former) and conjugate, without however giving riseto viable or fertile hybrids (Sonneborn 1974; Beale andPreer 2008). For either combination of mating types,40% of cells will form true conjugants in a mixed cultureof P. primaurelia and P. pentaurelia species, and the fre-quency rises to 90% when mating type E of P. tetraurelia ismixed with mating type O of P. octaurelia. Parameciumprimaurelia mating type E also weakly reacts with typeO of P. triaurelia. One would also expect a tendency for across-reaction between the closely related P. septaurelia andP. octaurelia or between P. septaurelia and P. tetraurelia.In fact, no such reactions are reported in the literature, butboth P. septaurelia and P. octaurelia can—depending

FIG. 2.—Unrooted NJ tree built by concatenating the nuclear loci GSPATG00000363001, GSPATG00027725001, and GSPATG00000546001(1,233 bp). Only nondiscordant strains are included in the analysis. Bootstrap values equal to or higher than 75 are shown.

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on their mating type—weakly react with P. primaurelia(Sonneborn 1975). It is worth noting that the genetic profileof most of the detected discordant strains corresponds tothat of P. primaurelia, which can cross-react with more spe-cies within the P. aurelia complex than any other, an ob-servation that remains unexplained.

We consistently find that the internal nodes of the mo-lecular phylogeny of the P. aurelia species complex are onlysupported by low bootstrap values. A similar poor phyloge-netic resolution has also been observed by previous studies(e.g., Coleman 2005; Hori et al. 2006; Barth et al. 2008). Thevirtual lack of phylogenetic resolution does not depend onthe type of marker examined, as neither nuclear nor mito-chondrial, and neither coding nor noncoding markers appearto be sufficiently informative. A plausible explanation forthe latter observation is that speciation in P. aurelia maynot have always proceeded as a branching process, followedby genetic isolation, but may have been the result of an ini-tial burst of speciation events, perhaps accompanied by ex-tensive hybridization. As the average divergence at silentsites between recent paralogs in P. tetraurelia is equal to2.17 (Aury et al. 2006)—.2 times higher than the maximumspecies divergence per silent site, we estimate in this study(0.9804, betweenP. tetraurelia andP. sonneborni)—our ob-servations indicate that the P. aurelia radiation postdates themost recent WGD and that speciation events may be associ-ated with differential retain of gene copies across isolatedpopulations (Lynch and Force 2000a).

Species Diversity and Geographic Distribution

Although our results do not suggest that huge globalpopulations of the P. aurelia species exist, we find the samealleles in strains isolated from multiple continents, consis-tent with regular (or recent) global dispersal of most of thespecies. These observations intriguingly suggest thatP. aurelia speciesmight bothhavea limited (effective)popu-lation size but also be cosmopolitan. In an attempt to providean explanation for this counterintuitive conclusion, we sug-gest that individualParamecia inhabiting relatively few anddurable habitats, which provide the long-term persistence ofthe species, would be dispersed to multiple geographicallyseparated (and often ephemeral) environments, generatingclonal populations whose life span is limited and dependson the features of the newly colonized habitats. The proposedscenario suggests a possibly cyclical and rapid transitionfrom limited geographic distribution to an effectively,temporary, and cosmopolitan distribution of Parameciumspecies, a dynamic that is similar to that proposed bysource–sink or source–pseudosink ecological models(Pulliam 1988; Watkinson and Sutherland 1995) and thatmay lead to a decrease in Ne (Whitlock and Barton 1997).

Additional and nonmutually exclusive explanationsfor allele sharing between strains inhabiting widely sepa-rated geographical areas would be strong selection againstmutations at silent sites and/or an exceptionally low muta-tion rate in Paramecium. These factors would influence theaccumulations of nucleotide changes after geographicalseparation of conspecific strains, and the extensive interspe-cific sequence divergence we observe would indicate fairlyold events of speciation in the P. aurelia species complex.

Whereas some support for selection against mutation at si-lent sites has been already provided (Salim et al. 2008), ourongoing study on the rate of spontaneous mutations in thegenome of P. tetraurelia will determine the likelihood ofthe low-mutation-rate hypothesis.

Conclusions

The average estimate of neutral genetic variability (ps)in a species is expected to reflect the global effective pop-ulation size of the species (Lynch 2006a). We found thatwithin the P. aurelia species complex, estimates of ps

are considerably higher when strains are grouped accordingto their reproductive compatibility but decrease greatlywhen the grouping criterion is the similarity of the strains’genetic profiles. This effect is due to the exclusion from theanalysis of a small set of strains with apparently aberrantmating behavior.

Overall, our study reveals that the average level of nu-clear genetic diversity in the free-living ciliate Parameciummay not be as exceptionally high as previously reported(Snoke et al. 2006) but is, on average, not as low as the de-gree of neutral genetic diversity reported for another ciliate,Tetrahymena thermophila (;0.003) (Katz et al. 2006). De-pending on the criteria that we use to tell true species apart,values of macronuclear ps within P. aurelia may be eithercomparable with or higher than the average diversity re-ported for a set of mostly pathogenic unicellular eukaryotes(0.0573 [SD 5 0.0777]) (Lynch 2006b) and for the unicel-lular green alga Chlamydomonas reinhardtii (0.032) (Smithand Lee 2008). Similar conclusions apply to the average ge-netic diversity observed at mitochondrial loci, where the ps

within the P. aurelia complex could be one of the highestvalues ever reported, together with P. multimicronucleatum(ps 5 0.308), a close outgroup of the P. aurelia complex(Snoke et al. 2006) or comparable with the ps of five otherunicellular (mostly pathogenic) eukaryotes (0.011 [SEM 50.004]) and C. reinhardtii (0.0085) (Smith and Lee 2008),after the removal of discordant strains.

Although this study cannot provide a definitive answerto the amount of genetic variability in Paramecium, our andprevious observations (Katz et al. 2006) seem to suggestthat levels of genetic diversity in free-living species maybe relatively low and comparable to those of pathogenictaxa. Our findings support the proposal that speciation inParamecium may not have always followed a cladistic pro-cess and highlight how the species problem in P. aurelia, anissue extensively addressed by Sonneborn during the lastcentury (Sonneborn 1937, 1957, 1974, 1975), may proveeven more complex than anticipated.

Supplementary Material

Supplementary tables 1-4, supplementary figure 1, andsupplementary material are available at Molecular Biologyand Evolution online (http://www.mbe.oxfordjournals.org/).

Acknowledgments

We thank T.G. Doak and C.L. McGrath for insightfulcomments and suggestions for the improvement of the




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http://www.mbe.oxfordjournals.org/


manuscript. We are grateful to E. Choi and V. Cloud for themaintenance of the Paramecium strains and to J.R. Preer Jrfor his patience and for sharing with us his own experienceand knowledge of Paramecium. We thank the RussianMinistry of Education and Science (project RNP2.2.3.1.4148) for supporting the maintenance of Collectionof Paramecium strains of the Laboratory of ProtozoanKaryology, St Petersburg State University, from whichoriginate most of the strains examined in this study. Thiswork was supported by the MetaCyte funding from theLilly Foundation to Indiana University.

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Laura Katz, Associate Editor

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Genetic Diversity in the Paramecium aurelia Species Complex

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