Molecular Ecology (2011) 20, 3644–3652 doi: 10.1111/j.1365-294X.2011.05150.x

Clonal genetic variation in a Wolbachia-infected asexualwasp: horizontal transmission or historical sex?

KEN KRAAIJEVELD,*† PADU FRANCO,* PETER D E KNIJFF ,† RICHARD STOUTHAMER‡ and

JACQUES J . M. V A N ALPHEN§

*Institute of Biology, Leiden University, PO Box 9505, 2300 RA Leiden, The Netherlands, †Department of Human Genetics,

Leiden University Medical Center S4-P, PO Box 9600, 2300 RC Leiden, The Netherlands, ‡Department of Entomology,

University of California, Riverside, CA 92521, USA, §IBED, University of Amsterdam, PO Box 94248, 1090 GE Amsterdam,

The Netherlands

Corresponde

E-mail: ken@

Abstract

Wolbachia are endocellular bacteria known for manipulating the reproductive systems of

many of their invertebrate hosts. Wolbachia are transmitted vertically from mother to

offspring. In addition, new infections result from horizontal transmission between

different host species. However, to what extent horizontal transmission plays a role in

the spread of a new infection through the host population is unknown. Here, we

investigate whether horizontal transmission of Wolbachia can explain clonal genetic

variation in natural populations of Leptopilina clavipes, a parasitoid wasp infected with a

parthenogenesis-inducing Wolbachia. We assessed variance of markers on the nuclear,

mitochondrial and Wolbachia genomes. The nuclear and mitochondrial markers

displayed significant and congruent variation among thelytokous wasp lineages,

showing that multiple lineages have become infected with Wolbachia. The alternative

hypothesis in which a single female became infected, the daughters of which mated with

males (thus introducing nuclear genetic variance) cannot account for the presence of

concordant variance in mtDNA. All Wolbachia markers, including the hypervariable wspgene, were invariant, suggesting that only a single strain of Wolbachia is involved. These

results show that Wolbachia has transferred horizontally to infect multiple female

lineages during the early spread through L. clavipes. Remarkably, multiple thelytokous

lineages have persisted side by side in the field for tens of thousands of generations.

Keywords: bacteria, evolution of sex, host parasite interactions, phylogeography

Received 9 February 2011; revision received 27 April 2011; accepted 3 May 2011

Introduction

Wolbachia are endocellular bacteria that infect a wide

range of invertebrates. Wolbachia are vertically transmit-

ted through egg cells and not through sperm cells, pre-

sumably because sperm cells contain too little

cytoplasm to harbour the bacteria. Male hosts are,

therefore, a dead end for Wolbachia. In many hosts, Wol-

bachia manipulates the host’s reproduction to enhance

its own transmission (O’Neill et al. 1997). Manipulative

effects of Wolbachia include the following: parthenogen-

esis induction, male-killing, feminization and cytoplas-

nce: Ken Kraaijeveld, Fax: +31 71 526 8285;

kenkraaijeveld.nl

mic incompatibility. The first three bias the sex ratio of

the host towards females, while the fourth gives a

reproductive advantage to Wolbachia-infected females

over uninfected females.

In addition to vertical transmission, Wolbachia can

transmit horizontally to infect new host species. The

phylogenies of Wolbachia are often strongly discordant

with those of their hosts, suggesting horizontal trans-

mission (Werren et al. 1995; Schilthuizen & Stouthamer

1997; Vavre et al. 1999). Experiments have shown that

Wolbachia can indeed be induced to establish new infec-

tions when transferred to a new host (Grenier et al.

1998; Huigens et al. 2000, 2004). However, whether hor-

izontal transmission also plays a role in the early

spread of a Wolbachia in a new host is unknown.

� 2011 Blackwell Publishing Ltd

CLONA L GENETIC V ARI ATI ON 3645

Parthenogenesis induction by Wolbachia occurs in sev-

eral species of haplodiploid insects. In uninfected ha-

plodiploids, males develop from unfertilized haploid

eggs, while females develop from fertilized diploid eggs

(arrhenotoky). In some haplodiploid hosts, Wolbachia

causes diploidization of unfertilized eggs, which then

develop as homozygous diploid females (thelytoky).

For example, in the parasitoid wasp Leptopilina clavipes,

Wolbachia disrupts chromosomal segregation at the first

mitotic division after meiosis (Pannebakker et al.

2004a). Thus, what normally would have been two hap-

loid cells now become a single diploid cell.

The observation of clonal genetic variation in popu-

lations of several hymenopteran hosts infected with

PI-Wolbachia may indicate the occurrence of horizontal

transmission. All offspring of a female infected with a

PI-Wolbachia are genetically identical to their mother.

An infected lineage is therefore genetically homoge-

neous except for novel mutations. However, popula-

tions of several host species infected with a PI-

Wolbachia display considerable genetic variation

(Plantard et al. 1998; Pannebakker et al. 2004b). For

example, Pannebakker et al. (2004b) found consider-

able nuclear genetic (AFLP) variation among thelytok-

ous L. clavipes lineages. This finding may indicate that

Wolbachia has transferred horizontally to infect multi-

ple, genetically distinct female lineages during the

early stages of its spread through the L. clavipes popu-

lation.

However, there are two additional explanations for

genetic diversity in thelytokous hosts, which make dif-

ferent predictions regarding the genetic variation to be

found in different components of the genome. Under

horizontal transmission of Wolbachia, both nuclear DNA

and mtDNA are expected to be variable among thely-

tokous lineages. Alternatively, a single female may have

become infected with Wolbachia and her infected daugh-

ters then mated with males (Stouthamer et al. 2010).

This would have introduced nuclear genetic variation

into the clonal lineage. Under this scenario, mitochon-

drial DNA (mtDNA) would be invariant, because this is

maternally transmitted. A third possibility is that multi-

ple females became infected independently by different

strains of Wolbachia. This would result in variance at

nuclear, mtDNA and Wolbachia DNA levels. A fourth

possibility, namely that infected females occasionally

reproduce sexually (e.g. Stone et al. 2008), can be ruled

out: Females from thelytokous lineages have nonfunc-

tional spermatheca and are unable to fertilize their eggs

(Kraaijeveld et al. 2009).

To test whether nuclear genetic variance in L. clavipes

is the result of horizontal transmission of Wolbachia, his-

torical sex or independent infections with Wolbachia, we

assessed genetic variation at all these levels. As neither

� 2011 Blackwell Publishing Ltd

the wasp lineages studied by Pannebakker et al.

(2004b), nor their DNA, were available, we collected a

new set of arrhenotokous and thelytokous lineages from

the field. We sequenced 1352 bp of mtDNA to evaluate

whether multiple maternal lineages explain the nuclear

variance. We used microsatellites to determine the level

of nuclear genetic variance among these lineages. Last,

we sequenced six Wolbachia genes to assess whether dif-

ferent wasp lineages are infected with distinct Wolbachia

strains.

Materials and methods

Wasp lineages

Leptopilina clavipes is a larval parasitoid of fungus-breed-

ing Drosophila. We obtained 25 gravid thelytokous

females from the Netherlands and 10 gravid arrheno-

tokous females from Spain (Table 1). In the Nether-

lands, females were either caught in the field or

cultured from stinkhorn fungus (Phallus impudicus). In

Spain, females were cultured from traps baited with

banana [see Pannebakker et al. (2004b) for further

details on collection methodology]. Each female was

used to initiate an isofemale lineage in the laboratory.

These were subsequently maintained by propagating

three females from each lineage each generation (see

Kraaijeveld et al. 2009 for details).

DNA was extracted from single females from each of

the isofemale lineage using the DNeasy blood and tis-

sue kit (Qiagen). The isofemale lineage had been cul-

tured in the laboratory for 10–50 generations before

DNA extraction for this study. This will probably have

resulted in the loss of genetic variation in the arrheno-

tokous lineages. Furthermore, all thelytokous lineages

were fully homozygous because of gamete duplication

by Wolbachia (Pannebakker et al. 2004a). This precluded

any population genetic estimates that are based on mea-

sured heterozygosity in the sampled population, such

as Fis.

Mitochondrial DNA

We amplified 1027 bp of the mitochondrial gene cyto-

chrome oxidase subunit 1 (CO1) in two fragments using

the primers and PCR conditions described by Scheffer

& Grissell (2003), except the reverse primer for the first

fragment, which was redesigned for this study:

5¢-TCATCTAAAAATTTTAATCCCAGT-3¢. We also ampli-

fied 325 bp of the ND1 gene using the primers F

5¢-ACTAATTCAGATTCTCCTTCT-3¢ and R 5¢-CAAC-

CTTTTAGTGATGC-3¢ (Smith & Kambhampati 1999)

using a standard PCR protocol at 50 �C annealing tem-

perature. Amplicons were sequenced at Macrogen.

Table 1 Collection details for the Leptopilina clavipes strains used in this study

Code Site Country Location Co-ordinates Date

Thelytokous trains

06STP (1) De Stulp NL Lage Vuursche N52�11.05¢E05�13.98¢

29 ⁄ 06 ⁄ 06

AR1 (2) Annanina’s rust NL Hilvarenbeek N51�29.18¢E05�09.28¢

05 ⁄ 09 ⁄ 06

AR2a (2) Annanina’s rust NL Hilvarenbeek N51�29.18¢E05�09.28¢

05 ⁄ 09 ⁄ 06

AR3a (2) Annanina’s rust NL Hilvarenbeek N51� 29.18¢E05�09.28¢

05 ⁄ 09 ⁄ 06

Aust2a (3) Austerlitz NL Austerlitz N52�04.43¢E05�18.53¢

04 ⁄ 09 ⁄ 06

BB1 (4) Bergherbos NL s’Heerenbergh N51�54.30¢E06�13.01¢

18 ⁄ 08 ⁄ 06

CDB1a (5) Cardanusbossen NL Doorwerth N51�58.48¢E05�48.22¢

04 ⁄ 09 ⁄ 06

CDB2 (5) Cardanusbossen NL Doorwerth N 51� 58.48¢E 05� 48.22¢

04 ⁄ 09 ⁄ 06

DB (6) Drakenburgh NL Baarn N52�13.02¢E05�16.98¢

08 ⁄ 07 ⁄ 05

DFW2 (7) Drents-Friese Wold NL Diever N52�53.41¢E06�18.11¢

30 ⁄ 08 ⁄ 06

GBW* (8) Groot Buunderkamp NL Wolfheze N52�00.00¢E05�46.98¢

15 ⁄ 06 ⁄ 00

Heikant (9) Heikant NL Oisterwijks N51�34.28¢E05�12.53¢

11 ⁄ 07 ⁄ 06

HTW(10) Huys te Warmond NL Warmond N52�12.00¢E04�30.00¢

21 ⁄ 08 ⁄ 06

KBH* (11) Klein Beekermark NL s’Heerenbergh N51�55.38¢E06�12.95¢

14 ⁄ 06 ⁄ 00

LD2-1 (12) De Lappendeken NL De Steeg N52�01.58¢E06�03.09¢

19 ⁄ 09 ⁄ 06

MGS3a (13) Allemansven NL Moergestel N51�32.49¢E05�12.20¢

25 ⁄ 08 ⁄ 06

MGS4e (13) Allemansven NL Moergestel N51�32.49¢E05�12.20¢

25 ⁄ 08 ⁄ 06

Rov1 (14) Roovert NL Tilburg N51�28.22¢E05�04.50¢

25 ⁄ 08 ⁄ 06

Rov3b (14) Roovert NL Tilburg N51�28.22¢E05�04.50¢

25 ⁄ 08 ⁄ 06

STP (1) De Stulp NL Lage Vuursche N52�11.05¢E05�13.98¢

08 ⁄ 07 ⁄ 05

WB1a (15) Warnsborn NL Schaarsbergen N52�01.16¢E05�51.23¢

19 ⁄ 09 ⁄ 06

WB3 (15) Warnsborn NL Schaarsbergen N 52� 01.16¢E 05� 51.23¢

19 ⁄ 09 ⁄ 06

ZH2 (16) De Horsten NL Wassenaar N52�08.09¢E04�24.45¢

14 ⁄ 08 ⁄ 06

ZH3-1 (16) De Horsten NL Wassenaar N 52� 08.09¢E 04� 24.45¢

14 ⁄ 08 ⁄ 06

ZH3-2 (16) De Horsten NL Wassenaar N52�08.09¢E04�24.45¢

14 ⁄ 08 ⁄ 06

Arrhenotokous strains

CB Can Bancells E Calonge N41�54.63¢E03�01.83¢

04 ⁄ 05 ⁄ 05

CBY Cabanyes E Calonge N41�50.81¢E03�02.72¢

04 ⁄ 05 ⁄ 05

3646 K. KRAAI JEVELD ET AL.

� 2011 Blackwell Publishing Ltd

Table 1 (Continued)

Code Site Country Location Co-ordinates Date

DC* Duna Continental E El Montgri N42�04.84¢E03�08.66¢

12 ⁄ 05 ⁄ 00

EJ El Jone E Calonge N41�52.64¢E03�04.60¢

04 ⁄ 05 ⁄ 05

EPG El Pou del Glac E Calonge N41�55.87¢E03�01.73¢

04 ⁄ 05 ⁄ 05

Mol1 Molli d’en Llambi E Llagostera N41�50.59¢E02�56.46¢

03 ⁄ 05 ⁄ 05

MS Mas Santet E Calonge N41�52.79¢E03�04.59¢

04 ⁄ 05 ⁄ 05

PdA Puig de l’Avellana E Sant Sadurni de l’Heura N41�56.55¢E02�59.36¢

03 ⁄ 05 ⁄ 05

PlB Puig de la Batteria E Calonge N41�53.85¢E03�03.56¢

03 ⁄ 05 ⁄ 05

TL Torre Lloreta E Calonge N41�52.19¢E03�04.84¢

02 ⁄ 05 ⁄ 05

Strains indicated with an * were the same as in Pannebakker et al. (2004b). Numbers in brackets correspond to those in Fig. S1

(Supporting information).

CLONA L GENETIC V ARI ATI ON 3647

For phylogenetic analysis, we aligned the sequences for

CO1 and ND1 without gaps using Falign as implemented

in eBioX (http://www.ebioinformatics.org). Haplotype

diversity and nucleotide diversity indices were calculated

using DNASP4 (Librado & Rozas 2009). The sequences

for the two mitochondrial genes were then concatenated,

and a matrix consisting only of the 10 polymorphic sites

was used as input for constructing a median-joining net-

work (Bandelt et al. 1999) with Network v. 4.516 (http://

www.fluxus- technology.com ⁄ sharenet.htm).

The genetic differentiation between thelytokous and

arrhenotokous lineages was assessed using the Snn met-

ric of Hudson (2000) and tested for statistical signifi-

cance using a permutation test with 1000 replicates.

Microsatellite diversity

We commissioned a genomic library enriched for three

types of tandem repeats (CA, GA and ATG; Genetic

Identification Services). Sequences were obtained for

100 insert-containing clones. We designed primers for

47 of these, from which we selected 16 polymorphic loci

for genotyping (Table S1, Supporting information).

We genotyped all individuals at 16 microsatellite loci.

Polymerase chain reactions (PCR) were performed

using standard conditions (all Ta = 55 �C). Forward

primers were labelled with a fluorescent dye according

to the protocol described by Li et al. (2007), and PCR

fragment sizes were visualized by electrophoresis on a

MegaBase 1000 sequencer (Amersham). Genotype pro-

files were scored manually.

A total of 10 arrhenotokous lineages and 25 thely-

tokous lineages were genotyped. Thelytokous geno-

� 2011 Blackwell Publishing Ltd

types were homozygous because of gamete

duplication by Wolbachia. All but two arrhenotokous

genotypes were homozygous, presumably because of

prolonged inbreeding in the laboratory. These two

heterozygous genotypes (one locus each) were artifi-

cially converted to homozygous by randomly selecting

one of the two alleles. The microsatellite data for both

thelytokous and arrhenotokous lineages were analysed

as if haploid.

Metrics of microsatellite diversity and differentiation

were calculated using GenAlex (Peakall & Smouse

2006). For each microsatellite locus, we counted the

number of alleles and the haploid genetic diversity h,

which estimates the probability that two individuals

will be different. The degree of subdivision among the

L. clavipes lineages was assessed using a principal com-

ponent analysis (PCA). The divergence between thely-

tokous and arrhenotokous lineages was assessed using

AMOVA. Network v. 4.516 was used to construct a med-

ian-joining halpotype network.

Comparison between mtDNA and microsatellitedivergence

We assessed whether the variance in mtDNA corre-

sponded to that in nuclear DNA in the thelytokous lin-

eages. We constructed pairwise matrices for the number

of nucleotide differences in the concatenated mtDNA

sequences and the haploid genetic distance using

DNASP4 and GenAlex, respectively. These two matrices

were then compared using a Mantel test from the

ecodist package in R 2.12.0 (R Development Core Team

2010).

3648 K. KRAAI JEVELD ET AL.

Wolbachia MLST

For each genetically distinct L. clavipes lineage, we

sequenced five Wolbachia housekeeping genes that are

standardly used for multilocus sequence typing (MLST:

coxA, hcpA, ftsZ, fbpA, gatb, Baldo et al. 2006). For each

of these five loci, specific primers for both A- and

B-type Wolbachia were tested. In addition to these rela-

tively slow-evolving genes, we also sequenced the hy-

pervariable Wolbachia surface protein (wsp) gene.

Wolbachia genes were amplified using the same tem-

plate DNA as the mtDNA PCRs. Primer sequences and

PCR conditions are described by Reumer et al. (2010).

Results

mtDNA

We found five distinct mtDNA haplotypes among the

thelytokous lineages and four mtDNA haplotypes

among the arrhenotokous lineages (Fig. 1). The nucleo-

tide diversity among the 25 thelytokous lineages

(p = 0.00045, haplotype diversity Hd = 0.3) was lower

than among the 10 arrhenotokous lineages (p = 0.00097,

haplotype diversity Hd = 0.53).

Most of this diversity was attributed to variation in

the CO1 locus. Of the 9 nucleotide polymorphisms at

this locus in the total data set, seven were synonymous

and two were nonsynonymous. The sequences for ND1

were identical for all lineages, except for a single non-

synonymous substitution that was fixed between thely-

tokous and arrhenotokous lineages.

The mtDNA sequences showed significant differentia-

tion between thelytokous and arrhenotokous lineages

(Snn = 0.97, P < 0.001). The average number of nucleo-

tide differences between the thelytokous lineages and

the arrhenotokous lineages for the concatenated CO1

Thelytokous Arrhenotokous

Fig. 1 Haplotype network based on the concatenated 1371-bp

mitochondrial COI ⁄ ND1 sequence. The sizes of the circles cor-

respond to the haplotype frequency, and branch lengths are

proportional to the genetic distance. Each haplotype is colour

coded.

and ND1 sequences was 3.42 (differences per site: Nei’s

Dxy = 0.00253).

The mtDNA data can be used to estimate the diver-

gence time of the thelytokous and arrhenotokous lin-

eages. Unfortunately, no fossil records are available for

Leptopilina clavipes to calibrate mtDNA mutation rate.

Raychoudhury et al. (2010) provided two estimates for

the synonymous mutation rate of CO1 in the parasitoid

wasp Nasonia: 7.4 · 10)5 and 2.0 · 10)5 per generation

for 92 synonymous sites or 8.0 · 10)7 and 2.2 · 10)7 per

base per generation, respectively. The thelytokous and

arrhenotokous L. clavipes lineages differed by on average

2.24 synonymous substitutions over 233.3 synonymous

CO1 sites or 9.6 · 10)3 substitution ⁄ base. Based on these

estimates, the thelytokous and arrhenotokous lineages

would have diverged 12 000 to 43 000 generations ago.

Microsatellites

Consistent with the results of a previous AFLP study

(Pannebakker et al. 2004b), we found considerable nuclear

genetic variance among the thelytokous wasp lineages

(Table 2, Fig. 2). Eleven distinct genotypes could be dis-

criminated among the 25 thelytokous wasp lineages.

The microsatellite genotypic variance was divided into

two distinct groups of clonal lineages (Figs 2 and 3).

The PCA analysis divided the microsatellite genotypes

into three distinct clusters: one cluster comprised of all

arrhenotokous lineages and two separate thelytokous

clusters (Fig. 3). Again, this is consistent with the AFLP

results described by Pannebakker et al. (2004b), who

termed the two thelytokous clusters T1 and T2. Two of

the thelyotokous lineages used in our study (GBW and

KBH) were originally collected by Pannebakker et al.

(2004b). Both belonged to the T1 AFLP group and had

very similar microsatellite genotypes. This confirms that

the large cluster of thelyotokous microsatellite geno-

types corresponds to the largest AFLP group. However,

we cannot be certain that the smaller group of diver-

gent microsatellite genotypes corresponds to the T2

AFLP group. The most common group of thelytokous

lineages was represented in 22 of 25 thelytokous lin-

eages, with the minority group only found in three lin-

eages (Fig. 1). Pannebakker et al. (2004b) found a

slightly less-skewed distribution of the two thelytokous

groups (T1 = 11 and T2 = 5 of 16 lineages, respectively).

The genetically distinct thelytokous lineages live side

by side in the field. Multiple females were obtained from

seven sites (five sets of two females and two of three

females; all except one set collected in the same year).

These females were collected from fungi growing within

a few hundred metres of each other, often on the same

day. In three of these cases, we found two distinct geno-

types at the same site and in one case three genotypes.

� 2011 Blackwell Publishing Ltd

Table 2 Summary statistics for the microsatellite data

Locus Na thelytokous Na arrhenotokous h thelytokous h arrhenotokous

E108 2 3 0.33 0.64

F2 4 5 0.50 0.60

F5 2 8 0.15 0.86

F101 8 8 0.77 0.86

F103 4 5 0.36 0.64

F118 3 3 0.35 0.34

F107 1 2 0.00 0.48

F102 5 4 0.34 0.48

F124 2 5 0.16 0.78

G10 2 2 0.15 0.50

E101 2 2 0.32 0.18

F129 4 5 0.39 0.78

E3a 2 3 0.32 0.66

F112 3 1 0.16 0.00

F130 3 4 0.39 0.72

F119 1 2 0.00 0.18

Mean ± SE 3.00 ± 0.44 3.88 ± 0.52 0.29 ± 0.05 0.54 ± 0.06

Na, number of alleles; h, haploid genetic diversity.

Thelytokous Arrhenotokous

Fig. 2 Haplotype network based on the homozygous microsat-

ellite genotypes. The sizes of the circles correspond to the

genotype frequency, and branch lengths are proportional to

the genetic distance. The colours of the circles correspond to

the mitochondrial haplotypes in Fig. 1.

Thelytokous

Coordinate 1

Coo

rdin

ate

2 Arrhenotokous

Fig. 3 Principal co-ordinate plot of the microsatellite geno-

types. The first two principal co-ordinates together explained

71.43% of the microsatellite variation. The colours correspond

to the mtDNA haplotypes in (Fig. 1).

CLONA L GENETIC V ARI ATI ON 3649

The thelytokous lineages were genetically distinct

from the uninfected, arrhenotokous lineages from

Spain (AMOVA /pt = 0.52, P = 0.001). The thelytokous ⁄arrhenotokous divergence explained 51% of the micro-

satellite variance.

Comparison between mtDNA and microsatellitedivergence

In the thelytokous lineages, variation in mtDNA

sequences corresponded to that in microsatellite geno-

type (Fig. 2). The number of mtDNA nucleotide differ-

� 2011 Blackwell Publishing Ltd

ences between lineages correlated with the pairwise

genetic distance estimated from the microsatellite data

(Mantel test r = 0.62, P = 0.002, Tables S2 and S3, Sup-

porting information). Lineages that were distinct from

other lineages in microsatellite genotype were thus also

different in their mtDNA sequence. Microsatellite and

mtDNA genetic distances were not significantly corre-

lated in the arrhenotokous lineages (Mantel test

r = )0.29, P = 0.18).

Wolbachia MLST

Sequences for all Wolbachia genes, including the hyper-

variable wsp locus, were identical in all thelytokous

3650 K. KRAAI JEVELD ET AL.

wasp lineages. There was thus no evidence that geneti-

cally distinct thelytokous lineages of L. clavipes were

infected with different strains on Wolbachia. Note, how-

ever, that the mutation rate of the Wolbachia genes

sequenced for this study is considerably lower than that

of, for example, CO1. Even the mutation rate of the wsp

gene, which is one of the most polymorphic regions in

the Wolbachia genome (Baldo et al. 2005), is probably

lower than that of CO1 (Raychoudhury et al. 2009).

Discussion

We found genetic variation among thelytokous lineages

of Leptopilina clavipes in mtDNA and microsatellite

(nuclear) DNA, but not in Wolbachia DNA. Moreover,

the mtDNA variation correlated with the nuclear DNA

variation. These results show that multiple female lin-

eages became infected with the same Wolbachia lineage.

The most likely explanation for genetic variation among

thelytokous lineages in this species is thus horizontal

transmission of Wolbachia during the early stages of

infection.

While previous studies have convincingly demon-

strated widespread interspecific horizontal transmis-

sion of Wolbachia, our study is the first to show that

horizontal transmission also plays a role in the spread

of a Wolbachia through a newly infected host popula-

tion. The most straightforward way in which horizon-

tal transmission could be achieved in L. clavipes is

through superparasitism, in which a single Drosophila

larva is parasitized by both a Wolbachia-infected and

an uninfected L. clavipes female. Superparasitism by

L. clavipes is common in the field (Driessen & Hemerik

1991), and experiments in Trichogramma have shown

that superparasitism can result in stable horizontal

intraspecific transmission in parasitoids (Huigens et al.

2000, 2004).

Our results cannot be explained by occasional sex by

thelytokous L. clavipes females. PI-Wolbachia-infected

females of Trichogramma are still able to fertilize their

eggs (Stouthamer & Kazmer 1994), thus introducing

nuclear (but not mitochondrial) genetic variation into

the thelytokous lineage. By contrast, L. clavipes females

are no longer able to utilize sperm because of their

degenerated spermatheca (Pannebakker et al. 2005; Kra-

aijeveld et al. 2009), ruling out contemporary sexual

generations in thelytokous lineages of this species.

However, this loss of sexual functionality can only have

originated after the spread of the Wolbachia infection

(and may have been selected for during the spread of

Wolbachia through the host population; Stouthamer

et al. 2010). It is possible that thelytokous L. clavipes

females were still able to reproduce sexually during the

early stages of the spread of Wolbachia. Historical sexual

reproduction by thelytokous females may account for

some of the nuclear genetic variance seen among the

thelytokous lineages but cannot explain the mtDNA

variation we found.

A similar pattern of concordant variation in nuclear

DNA and mtDNA in thelytokous L. clavipes lineages

would be expected if multiple L. clavipes lineages

became infected independently with different Wolbachia.

In that case, however, the different thelytokous lineages

should also be associated with different Wolbachia geno-

types. We found no evidence for such a pattern,

because all Wolbachia sequences we obtained were iden-

tical. Note, however, that the Wolbachia markers we

used evolve more slowly than the mitochondrial and

nuclear markers. These markers are thus not variable

enough to detect small differences between closely

related Wolbachia strains.

In general, genetic variation in asexual organisms is

expected to result from multiple origins from asexual

ancestors, rather than mutation accumulation (Vrijen-

hoek 1998). However, novel mutations may contribute

to small differences between thelytokous lineages, espe-

cially in hypervariable markers such as microsatellites.

The larger differences between most divergent thelytok-

ous lineages seen in our study are similar to the differ-

ence between the thelytokous and arrhenotokous

lineages. Differences of this magnitude could only be

explained by mutation accumulation if the northern lin-

eages became infected with Wolbachia and switched to

thelytokous reproduction very soon after diverging

from the Spanish population. We know this was not the

case. Experiments by Kraaijeveld et al. (2009) showed

that males derived from thelytokous lineages displayed

a mate preference for thelytokous females, while arrhe-

notokous males preferred arrhenotokous females. This

assortative mating can only be explained by divergence

during a time when males from both populations were

still present in the field. Thus, the ancestors of the

thelytokous and arrhenotokous lineages diverged as iso-

lated arrhenotokous populations for a considerable per-

iod of time, leaving little time for mutation

accumulation after Wolbachia became established.

A remarkable feature of our results is that the genet-

ically distinct thelytokous lineages live side by side in

the field. In general, selection (ecological or frequency

dependent) is thought to be required for the mainte-

nance of clonal variation in the field (Jokela et al.

2003). In the absence of such selection, genetic drift

would be expected to erode genetic variation (Simon

et al. 2003). We collected up to three distinct thelytok-

ous lineages from the same small forest patch, some-

times from fungi that were within metres of each

other, during the same field trips. Habitat, feeding

substrate and phenology were identical for these lin-

� 2011 Blackwell Publishing Ltd

CLONA L GENETIC V ARI ATI ON 3651

eages. It is possible that these lineages specialize on

different Drosophila hosts, but we have no indication

that they perform differently when offered Drosophila

phalerata in the laboratory, which is the main host of

L. clavipes in the field. The different thelytokous lin-

eages thus appear to be ecologically equivalent. Per-

haps, the genetic variation seen in thelytokous

L. clavipes populations is transient and will eventually

disappear with the (local) extinction of the less com-

mon lineages. Alternatively, the co-existence of multi-

ple thelytokous lineages may persist when clonal

competition is low. The substrate used by L. clavipes,

stinkhorn fungi, is patchily distributed and ephemeral.

Driessen & Hemerik (1991) showed that the wasps are

distributed over substrate patches in an aggregated

fashion, perhaps indicating that the effective overlap

in resource use by different clones is small.

Our results confirm the findings by Pannebakker

et al. (2004b) in that the thelytokous lineages are geneti-

cally distinct from the arrhenotokous lineages in Spain.

Our mtDNA results suggest that these two populations

diverged between 12 000 and 43 000 generations ago.

Assuming two generations per year, this would place

the divergence between 6000 and 21 500 years ago or

late Pleistocene ⁄ early Holocene. This was a time of cli-

matic instability, which included the cold phases of the

last glacial maximum and the Younger Dryas, as well

as warm episodes (Frenzel et al. 1992). It is possible that

the European L. clavipes population was divided into

separate refugia during these cold phases, for example

in Iberia and the Balkans (Pannebakker 2004), but we

cannot rule out other scenarios. Unfortunately, recent

attempts to collect this species in Italy, the Balkans and

Greece were unsuccessful (K. Kraaijeveld, unpublished).

Furthermore, mtDNA substitution rates may vary

between species, which would bias our estimate of

divergent time. Pinpointing the exact time and cause of

the divergence between the Spanish and Northern

European populations is thus not possible with current

data.

In summary, females of the northern lineages of

L. clavipes became infected with Wolbachia after having

been separated from the Spanish lineages for a consid-

erable period. Wolbachia then spread to fixation

through horizontal as well as vertical transmission.

The thelytokous lineages appear not to have spread

into south-west France (Pannebakker et al. 2004b) and

therefore have not come in contact with the uninfected

Spanish wasps.

Acknowledgements

We thank Kees Koops for help with the culturing, Barbara

Reumer, Rob Kraaijeveld and Femmie Kraaijeveld-Smit for

� 2011 Blackwell Publishing Ltd

help with the fieldwork. This work was supported by a Veni

grant from NWO to KK.

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K.K’s research focusses on the infection dynamics of partheno-

genesis-inducing Wolbachia and their consequences for host

evolution. P.F. currently runs the wildlife conservation society

program Colombia. This paper is part of his MSc research. P.K.

is a professor of human genetics, specializing in population

genetics. R.S. is a professor of entomology whose research has

focused on the host-symbiont relationship between partheno-

genesis inducing Wolbachia and parasitoid wasps. J.A. uses

molecular tools to study population structure in amphibians

and insect parasitoids.

Data accessibility

DNA sequences: GenBank accessions HM999658-HM999666

(CO1), HM999649-HM999650 (ND1), HM999630-HM999648

(microsatellite loci) and HM999651-HM999656 (Wolbachia).

Wolbachia MLST gene sequences: Wolbachia MLST database

(http://pubmlst.org/wolbachia/ under mlst id = 342 and wsp

id = 547).

Microsatellite data: DRYAD entry doi: 10.5061/dryad.hh1f7.

Supporting information

Additional supporting information may be found in the online

version of this article.

Table S1 Primers used for microsatellite genotyping (all

5¢—3¢).

Table S2 Matrix of pairwise haploid genetic distance between

thelytokous L. clavipes lineages estimated using the microsatel-

lite data.

Table S3 Pairwise matrix of the number of nucleotide differ-

ences in mtDNA sequence between thelytokous L. clavipes lin-

eages.

Fig. S1 Map of collection sites of Leptopilina clavipes in The

Netherlands. Numbers correspond to those in Table 1.

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