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Genetics of the Pig Tapeworm in Madagascar Reveal a History of Human Dispersal and Colonization Tetsuya Yanagida1*¤, Jean-François Carod2, Yasuhito Sako1, Minoru Nakao1, Eric P. Hoberg3, Akira Ito1

1 Department of Parasitology, Asahikawa Medical University, Asahikawa, Hokkaido, Japan, 2 Institut Pasteur de Madagascar, Antananarivo, Madagascar, 3 US Department

of Agriculture, Agricultural Research Service, US National Parasite Collection, Animal Parasitic Diseases Laboratory, Beltsville, Maryland, United States of America

Abstract

An intricate history of human dispersal and geographic colonization has strongly affected the distribution of human pathogens. The pig tapeworm Taenia solium occurs throughout the world as the causative agent of cysticercosis, one of the most serious neglected tropical diseases. Discrete genetic lineages of T. solium in Asia and Africa/Latin America are geographically disjunct; only in Madagascar are they sympatric. Linguistic, archaeological and genetic evidence has indicated that the people in Madagascar have mixed ancestry from Island Southeast Asia and East Africa. Hence, anthropogenic introduction of the tapeworm from Southeast Asia and Africa had been postulated. This study shows that the major mitochondrial haplotype of T. solium in Madagascar is closely related to those from the Indian Subcontinent. Parasitological evidence presented here, and human genetics previously reported, support the hypothesis of an Indian influence on Malagasy culture coinciding with periods of early human migration onto the island. We also found evidence of nuclear-mitochondrial discordance in single tapeworms, indicating unexpected cross-fertilization between the two lineages of T. solium. Analyses of genetic and geographic populations of T. solium in Madagascar will shed light on apparently rapid evolution of this organism driven by recent (,2,000 yr) human migrations, following tens of thousands of years of geographic isolation.

Citation: Yanagida T, Carod J-F, Sako Y, Nakao M, Hoberg EP, et al. (2014) Genetics of the Pig Tapeworm in Madagascar Reveal a History of Human Dispersal and Colonization. PLoS ONE 9(10): e109002. doi:10.1371/journal.pone.0109002

Editor: Yong-Gang Yao, Kunming Institute of Zoology, Chinese Academy of Sciences, China

Received June 5, 2014; Accepted September 6, 2014; Published October 15, 2014

This is an open-access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All nucleotide sequence files are available from GenBank (accession numbers AB781355-AB781369).

Funding: This study was supported by the Institut (http://www.pasteur.mg/) Pasteur de Madagascar and by the Japan Society for Promotion of Science (JSPS: http://www.jsps.go.jp/) Asia/Africa Scientific platform (2006-2011), the Grant-in-Aid for Scientific Research from JSPS (21256003, 24256002) and the Special Coordination Fund for Promoting Science and Technology from the Ministry of Education, Japan (2010-2012) to A. Ito. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* Email: [email protected]

¤ Current address: Laboratory of Veterinary Parasitology, Joint Faculty of Veterinary Medicine, Yamaguchi University, Yoshida, Yamaguchi, Japan

Introduction

The pig tapeworm Taenia solium (Cestoda: Taeniidae) is an etiologic agent of cysticercosis, an important zoonosis and

neglected tropical disease, and recently ranked as the most

important food-borne parasites on a global scale [1]. The lifecycle

of T. solium includes humans as the only definitive hosts and domestic pigs as principal intermediate hosts. Cysticercosis refers

to infection of various tissues of swine or humans with cysticerci

larvae due to ingestion of eggs released from people harboring

adult worms in the intestine. Cysticercosis of the central nervous

system (neurocysticercosis or NCC), warrants special attention

because it is a major cause of seizures and epilepsy in endemic

areas [2] and can be lethal especially in remote areas of developing

countries [3]. T. solium is distributed worldwide where local people consume pork without meat inspection. We previously

reported that T. solium can be divided into two mitochondrial (mtDNA) genetic linages, Asian and Afro-American which differ in

the clinical manifestations of human cysticercosis [4]. Their

distributions are geographically disjunct in Asia or Africa and

Latin America [4]. It has been postulated that T. solium emerged from Africa with early modern humans and through geographic

expansion became distributed initially across Eurasia prior to the

advent of agriculture and domestication of swine [5–7]. Phyloge-

netic studies have suggested that divergence of the two lineages

occurred in the Pleistocene [4,6,8]. Recently, sympatry of both

mitochondrial lineages was confirmed in Madagascar [6,8].

Madagascar is a country known to be hyper-endemic for

cysticercosis [9,10]. Cysticercosis in pigs results in condemnation

of carcasses, particularly in heavy infections, and thus constitutes a

considerable economic challenge. Understanding the current

distribution for these parasites and the historical factors involved

in geographic colonization of Madagascar can contribute insights

of importance in developing a capacity for control and mitigation

of infections in swine and human hosts.

Malagasy people are divided into 18 ethnic groups and have

diverse cultures. Surprisingly, the first human settlement occurred

approximately 2000 years ago as one endpoint of Austronesian

migration. Linguistic and archeological evidence suggests that the

Malagasy people have mixed ancestry from Island Southeast Asia

(ISEA), especially Borneo, and from East Africa [11]; dual origins

confirmed by analyses of mtDNA and nuclear DNA [12]. In

addition, a contribution to the gene pool of Malagasy people from

India has recently been suggested by mtDNA genetic analysis [13].

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Prehistoric human migrations can also be traced by parasitological

evidence. For example, archaeoparasitology of some intestinal

parasites have indicated the existence of human migration routes

into the New World other than those involving Bering Land

Bridge [14]. Phylogenetic analysis suggested that T. solium has been introduced into Madagascar multiple times from a number of

different areas [8], but the dynamics of these introductions and

establishment were not fully elucidated. In the present study,

reciprocal insights for the distributional history of hosts and

parasites emerge from an exploration of T. solium and human occupation of Madagascar.

Historically disjunct populations of T. solium are now in sympatry in Madagascar, affording a unique opportunity to

explore the possibility of cross-fertilization and hybridization as a

fundamental process among cestodes, and concurrently reflect on

the degree of isolation and distinct nature of these genotypes.

Cestodes are hermaphrodites with two potential modes of

reproduction, self- and cross-fertilization. T. solium has often been referred to as a self-fertilizer because it is nearly always found

alone in the human intestine. However, random amplified

polymorphic DNA showed heterozygosity in cysticerci of T. solium, suggesting cross-fertilization between different individual worms [15]. Consequently, it may be assumed that the two

genotypes of T. solium can cross-fertilize in infections involving multiple adults, which may occur early in the infection process.

Analysis of maternal inherited mtDNA alone, however, is not

sufficient to examine putative hybridization events. Thus, we

initially established nuclear DNA markers to differentiate

geographic variation in T. solium. Secondarily, genetic polymor- phism of T. solium in Madagascar was investigated to clarify whether hybridization occurs on the island.

Materials and Methods

Parasite isolates and DNA sequencing During 2005 to 2008, 57 pigs slaughtered from 16 different

localities in 5 provinces on Madagascar were found positive for T. solium cysticerci. No specific permissions were required for the field survey, and it did not involve endangered or protected

species. Meat inspectors in each province were requested to collect

infected pig meats at slaughterhouses from the various locations.

Pigs were regularly slaughtered at the official slaughterhouses of

each city (Table S1), and the slaughtering was controlled by meat

inspectors according to the regulations of the Republic of

Madagascar. Pigs were sacrificed for routine slaughterhouse

purposes and not for research purposes. When positive for Taenia cysticerci, infected meats were cut and inserted into sterile

containers, and sent to the Pasteur Institute of Madagascar within

24 hours. Then the cysticerci were extracted and washed at the

laboratory, and frozen at 220uC until use. All samples were then fixed with 70% ethanol and shipped to Japan according to the

research agreement between Pasteur Institute of Madagascar and

Asahikawa Medical University. One or two cysts from each pig

were subjected to molecular analysis. The genomic DNA of each

cyst was extracted by DNeasy blood and tissue kit (Qiagen), and

subsequently used as a template for polymerase chain reaction

(PCR). For the mtDNA gene markers, the entire cytochrome c oxidase subunit I (cox1) and cytochrome b (cob) were amplified by PCR using previously reported primer pairs [4]. PCR products

were treated with illustra ExoStar (GE Healthcare) to remove

excess primers and dNTPs, and directly sequenced with a BigDye

Terminator v3.1 and a 3500 DNA sequencer (Life Technologies).

Nuclear gene markers including RNA polymerase II second

largest subunit (rpb2), phosphoenolpyruvate carboxykinase (pepck),

DNA polymerase delta (pold) and a low-molecular-weight glycoprotein antigen (Ag2) were amplified using primer pairs published previously [16,17]. These nuclear genes were chosen

because they have been shown to be useful for the molecular

phylogeny of taeniid tapeworms including species of Taenia (rpb2, pepck and pold) or for differentiating geographic genotypes of T. solium (Ag2). Initially, 41 geographic isolates of T. solium from 14 countries were used to investigate the geographical variability of

nuclear gene markers. PCR products were sequenced with the

same protocols as mtDNA gene markers. When geographical

variations were found, new primers were designed to amplify the

short fragments including mutation sites in order to reduce the cost

and labor. PCR was performed in 20 mL volumes containing 0.5 units of Ex Taq Hot Start Version (TaKaRa, Japan), 0.2 mM of

dNTP, 16Ex Taq Buffer with a final MgCl2 concentration of 2.0 mM, 15 pmol of each primer and 1.0 mL of genomic DNA. PCR amplification consisted of initial denaturation of 94uC for 2 min, 35 cycles of 94uC for 15 sec, 55uC for 15 sec and 72uC for 30 sec, and a terminal extension at 72uC for 1 min. In cases of double peaks in the sequencing of nuclear genes, PCR products

were ligated into pGEM-T plasmid vector (Promega) and then

introduced into Escherichia coli DH5a. At least 10 clonal colonies were picked from an agar plate and their insert DNAs were

sequenced to confirm allelic polymorphism.

Data analysis Nucleotide sequences of the mitochondrial cob (1068 sites) and

cox1 (1620 sites) were concatenated into a total sequence (2688 sites). They were aligned by Clustal W 2.0 [18] with those

sequences available in public databases. Amino acid sequences

were inferred with reference to the echinoderm mitochondrial

genetic code [19]. Pairwise divergence values among the obtained

nucleotide sequences were calculated using the MEGA5 package

[20] using Kimura’s two parameter model with a c-shaped parameter (a = 0.5). The identification of mtDNA haplotypes and the drawing of their network was computed by TCS 1.2 software

[21] using statistical parsimony [22]. Evaluation of the rate of

outcrossing was based on an estimate of the inbreeding coefficient

for each nuclear locus and deviation from Hardy-Weinberg

proportions as F = 1-Hobs/Hexp, where H is the actual population heterozygosity and Hexp is the expected heterozygosity under H – W equilibrium.

Results

Mitochondrial DNA phylogeography In the present study, we collected 109 cysticerci larvae from 57

pigs across 5 provinces on Madagascar. In total, 8 haplotypes

(MDG1 to MDG8) of concatenated cox1 and cob genes were detected. When compared with individual genes, the numbers of

haplotypes were reduced to 3 (cob) and 7 (cox1). All the nucleotide sequences of each haplotype are deposited in GenBank with

accession numbers AB781355-AB781364. The frequency of the

nucleotide substitution was 1.6% (17 sites/1068 sites) in cob and 1.4% (22/1620) in cox1 (Tables S2 and S3). Among 39 point mutation sites identified, 24 (61.5%) were synonymous and 15

(38.5%) were non-synonymous substitutions. The maximum value

of divergence among the 8 haplotypes was 1.4%. Among the

mtDNA gene sequences of T. solium deposited in the public databases, 14 sets of the complete cob and cox1 gene sequences were concatenated and used for the haplotype network analysis

together with those from Madagascar (Table 1). These sequences

were chosen because they had unequivocal published references

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allowing confirmation that the sequences of the two genes were

obtained from one individual parasite.

Network analysis clearly showed these 8 haplotypes are divided

into two genotypes (Fig. 1). Six haplotypes (MDG1-6) were the

Asian genotype and the remaining two (MDG7-8) were the Afro-

American genotype. Overall, 77% (84/109) of the Madagascan

haplotypes were the Asian genotype (Table 2). The Asian

genotype was found in all examined provinces and was generally

dominant except in Toliara. The Afro-American genotype was

identified in 4 of 5 examined localities. Among the haplotypes

obtained, MDG1 was the major (62%), followed by MDG7 (23%).

Among 52 pigs in which two cysts were examined, the different

haplotypes were simultaneously obtained in 3 hosts; Asian and

Afro-American haplotypes (MDG1 and MDG7) were identified

from two hosts, and the different Asian haplotypes (MDG1 and

MDG4) were obtained from one host. MDG1 was 100% identical

to the haplotype obtained from a pig in Nepal [23], and one base

different from the Indian haplotype. All Asian haplotypes from

Madagascar are grouped with those from the Indian Subconti-

nent. On the other hand, these Asian haplotypes were distantly

related to that from Papua, Indonesia. Further, the cox1 haplotype of the isolate from Bali Island, Indonesia [23] was also distantly

related. In contrast, MDG7 was one base different from MDG8

and the haplotypes from Mexico, Ecuador, Bolivia.

Nuclear DNA Among the Ag2, rpb2 and pold locus, two (Ag2 and rpb2) or

three (pold) alleles were confirmed from the 14 geographical isolates from 14 countries; no geographical variation was found in

the pepck locus. Subsequently, Ag2, rpb2 and pold were chosen as appropriate nuclear DNA markers to discriminate the Asian and

Afro-American genotypes of T. solium. To amplify the target regions including the variable sites, new primers were designed for

rpb2 and pold (Table S4). An additional 27 geographical isolates were analyzed using these new primer sets, to confirm geograph-

ical variation. Ag2A, rpb2A and poldA (Asian alleles) were only found in Asia and Ag2B, rpb2B, poldB and poldC (Afro-American Alleles) were obtained from Latin American and African countries

(Table 3). The sequence difference among the alleles was 1–3 bp.

All the nucleotide sequences of each allele of rpb2 and pold are deposited in GenBank with accession numbers AB781365-

AB781369.

Establishment of nuclear DNA markers allowed us to investigate

possible hybridization events in Madagascar. All three nuclear

genes were amplified and sequenced for the same 109 cysts as

mtDNA genes. Overall, the Asian alleles were the majority in

Madagascar with frequencies of 0.81–0.84 (Table 2). Asian alleles

were the majority in all the examined regions except for Toliara,

and the frequencies of Afro-American alleles in the region were

0.60–0.72. No new alleles were identified among these three loci.

Among 12 cysts, the nucleotide sequences of one or more loci

could not be determined by direct sequencing because of double

Table 1. Mitochondrial haplotypes of T. solium used for the phylogeographic analysis.

Haplotypes a

Localities Accession numbers References

Cox1 Cob

MDG1 Madagascar AB781355 AB781362 This study

MDG2 Madagascar AB781356 Same as MDG1 This study

MDG3 Madagascar Same as MDG1 AB781363 This study

MDG4 Madagascar AB781357 Same as MDG1 This study

MDG5 Madagascar AB781358 Same as MDG1 This study

MDG6 Madagascar AB781359 Same as MDG1 This study

MDG7 Madagascar AB781360 AB781364 This study

MDG8 Madagascar AB781361 Same as MDG7 This study

CHN1 China AB066485 AB066570 Nakao et al. 2002 4

CHN2 China AB066486 AB066571 Nakao et al. 20024

ID-BA Bali, Indonesia AB631045 Not determined Swastika et al. 2012 24

ID-PA Papua, Indonesia AB066488 AB066573 Nakao et al. 2002 4

IND India AB066489 AB066574 Nakao et al. 20024

NPL1 Nepal AB491985 AB781746 Yanagida et al. 2010 23

NPL2 Nepal AB491986 Same as MDG1 Yanagida et al. 2010 23

THA Thailand AB066487 AB066572 Nakao et al. 20024

BRA Brazil AB066492 AB066577 Nakao et al. 2002 4

CMR Cameroon Same as MEX1 AB066579 Nakao et al. 2002 4

ECU Ecuador AB066491 AB066576 Nakao et al. 20024

MEX1 Mexico AB066490 AB066575 Nakao et al. 2002 4

MEX2 Mexico FN995657 FN995661 Michelet & Dauga 2012 6

MEX3 Mexico FH995658 FN995662 Michelet & Dauga 20126

TZA Tanzania AB066493 AB066578 Nakao et al. 2002 4

a The mitochondrial haplotypes were determined based on the concatenated nucleotide sequences of complete cox1 (1620 bp) and cob (1068 bp), except for ID-BA. doi:10.1371/journal.pone.0109002.t001

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Figure 1. Mitochondrial genotypes of T. solium in Madagascar. (A) Pie charts illustrating the frequencies of the Asian and Afro-American mitochondrial genotypes of T. solium in each collection site. The numbers in the charts show the sample size for parasite isolates examined. Madagascar is divided into the 7 former provinces. (B) The haplotype network of concatenated mtDNA gene sequences. The size of the ellipses is roughly proportional to the haplotype frequency, and the actual numbers of haplotypes (.1) are enclosed in parentheses. doi:10.1371/journal.pone.0109002.g001

Genetics of the Pig Tapeworm in Madagascar

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peaks in the sequence electropherograms. As the result of cloning

of the polymorphic PCR amplicons, two alleles were detected at

an approximate ratio of 1:1. These cases were considered to be

heterozygous in each locus. Two cysts obtained from one pig were

heterozygous at the all three loci examined. Twenty-two cysts

possessed discordant mitochondrial and nuclear genotypes, Asian

and Afro-American, in at least one nuclear locus (Table 4). The

inbreeding coefficient (F) was estimated only for the sub- population in Toliara because of the considerably biased allele

frequency in the other sub-populations; at this locality, F was equal to 0.86 (Ag2), 0.90 (rpb2) and 0.79 (pold).

Discussion

The sympatric distribution of Asian and Afro-American

mitochondrial genotypes was confirmed on Madagascar, corrob-

orating a prior report [6,8]. Although the Afro-American

mitochondrial genotype previously was identified only in Toliara

[8], we confirmed the co-occurrence of Asian and Afro-American

genotypes in 4 out of 7 provinces, indicating a widespread

distribution for the two mitochondrial genotypes across the island.

Major genotypes differed geographically and across provinces.

The Asian genotype was generally dominant at all localities except

in Toliara, where 64% of the parasite isolates were the Afro-

American genotype.

Differences in the distribution of the dominant genotypes of T. solium among provinces can be attributed to disparate history and ethnic origins in each region and patterns of human dispersal and

migration over the past several thousand years. Phylogenetic

analyses of Taenia have suggested a relatively deep origin in Africa for T. solium, which may have initially parasitized hominin ancestors of modern humans in the early Pleistocene following a

host-switching event from large carnivores [5,7,25]. It has been

postulated that T. solium emerged from Africa with early modern humans and through geographic expansion became distributed

initially across Eurasia prior to the domestication of swine which

now represent a primary intermediate host [5,6]. Although there is

no direct evidence, phylogenetic studies using mtDNA markers

have suggested the divergence of the two genotypes, now

associated respectively with Africa/America and with southern

Asia/Indian Subcontinent occurred in the Pleistocene [4,6,8].

The dominant haplotype in Madagascar (MDG1) demonstrates

Asian affinities and is genetically most similar to those from Nepal

and India, but distantly related to that from Papua, Indonesia.

Further, a cox1 gene sequence of the isolate from Bali Island [24] was distantly related to MDG1 and other haplotypes from

Madagascar. Consequently, it appears that the origin of the Asian

genotype on Madagascar is not from ISEA, coincidental with the

first human immigrants, but from the Indian Subcontinent.

Although Asian origins of the Malagasy people have generally

been linked to immigrants and populations from ISEA, our result

and recent report on human mitochondrial genetics [13] indicate

the importance of Indian influence on the diversity of people and

culture in Madagascar consistent with and reflecting a history of

human dispersal within the past 2,000 years.

On the other hand, the dominant Afro-American haplotype in

Madagascar (MDG7) is closely related to those from Mexico and

Ecuador. It does not imply a direct link for Madagascan and Latin

American populations, because it is apparent that Afro-American

haplotypes have been widely disseminated and the same haplotype

can be obtained from both African and Latin American countries

[4,8]. It was suggested that T. solium was introduced into Latin America from Europe or Africa coincidental with European

expansion and development of maritime trade routes after the

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Table 3. Distribution of alleles at each nuclear locus around the world.

Localities No. isolates examined Alleles

Ag2 rpb2 pold

China 4 Ag2A rpb2A poldA

Thailand 2 Ag2A rpb2A poldA

Papua, Indonesia 2 Ag2A rpb2A poldA

Nepal 3 Ag2A rpb2A poldA

India 4 Ag2A rpb2A poldA

Vietnam 1 Ag2A rpb2A poldA

Asian total 16

Tanzania 7 Ag2B rpb2B poldB

Mozambique 7 Ag2B rpb2B poldB

South Africa 2 Ag2B rpb2B poldC

Cameroon 4 Ag2B rpb2B poldC

Mexico 1 Ag2B rpb2B poldB

Ecuador 2 Ag2B rpb2B poldC

Peru 1 Ag2B rpb2B poldC

Brazil 1 Ag2B rpb2B poldC

Afro-American total 25

doi:10.1371/journal.pone.0109002.t003

Table 4. Genotypes of T. solium showing nuclear-mitochondrial discordance.

ID of samples MtDNA haplotype a Genotype at each locus

a,b Localities

Ag2 rpb2 pold

TsolMDG21b MDG1 B/B B/B A/A Toliara

TsolMDG29a MDG1 B/B A/A A/A Toamasina

TsolMDG62a MDG1 A/A A/A C/C Antananarivo

TsolMDG62b MDG1 A/A A/A A/C Antananarivo

TsolMDG67a MDG1 B/B B/B A/C Toliara

TsolMDG68b MDG1 B/B B/B C/C Toliara

TsolMDG04a MDG7 B/B A/B A/A Antananarivo

TsolMDG04b MDG7 B/B A/A A/A Antananarivo

TsolMDG12b MDG7 A/A A/A A/C Antananarivo

TsolMDG13a MDG7 A/B A/A A/A Antananarivo

TsolMDG13b MDG7 B/B A/A A/A Antananarivo

TsolMDG25a MDG7 A/B A/B A/C Toliara

TsolMDG25b MDG7 A/B A/B A/C Toliara

TsolMDG28a MDG7 A/A B/B C/C Toliara

TsolMDG37a MDG7 A/A A/A A/A Toamasina

TsolMDG37b MDG7 A/A A/A A/A Toamasina

TsolMDG50a MDG7 A/A A/B A/A Mahajanga

TsolMDG50b MDG7 A/A A/A A/C Mahajanga

TsolMDG21a MDG7 B/B B/B A/C Toliara

TsolMDG68a MDG7 B/B B/B A/C Toliara

TsolMDG69a MDG7 A/B B/B C/C Toliara

TsolMDG69b MDG7 B/B B/B A/C Toliara

a Haplotypes and alleles in bold are Afro-American ones. b

Genotypes with underline indicate those at heterozygous loci. doi:10.1371/journal.pone.0109002.t004

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15th century [4,26]. The dominance of the Afro-American

genotype at Toliara, where the current populace is primarily of

African descent, suggests that parasites were introduced to

Madagascar, probably recurrently, with people and swine from

coastal East Africa in a time frame within the past hundreds of

years, although clarification requires further study of isolated

populations in areas bordering the Mozambique Channel.

Both Asian and Afro-American genotypes on Madagascar

showed a simple network with the major (MDG1 and MDG7) and

satellite haplotypes. This result indicates a minimum of two

independent events of anthropogenic introduction for T. solium from historically disjunct geographic regions in relatively shallow

ecological time. It is not clear whether T. solium was introduced with infected pigs or humans, but it is reasonable to consider that

establishment occurred after the first human settlement 2000 years

ago because humans are the only definitive hosts. Phylogeography

of swine has revealed the distribution of different haplogroups

among South Asia, mainland Southeast Asia and ISEA, resulting

from Neolithic, human-mediated translocation [27,28]. Thus,

genetic analysis of the pigs in Madagascar may shed light on how

the tapeworm dispersed across the Indian Ocean.

In the present study, nuclear-mitochondrial discordance was

confirmed in all three loci examined, suggesting hybridization

between individual worms possessing different genotypes in the

recent past. Two cysts from a pig in Toliara were heterozygous at

all three loci, suggesting these were F1 hybrids between Asian and

Afro-American populations; this genotype could appear at the F2

or later generation by self-fertilization of a hybrid-derived

individual worm. Nuclear-mitochondrial discordance in T. solium has been confirmed only in Madagascar to date, indicating the

hybridization event occurred on the island. The inbreeding

coefficient F of the sub-population in Toliara was about 0.8–0.9. If F is interpreted as the rate of selfing [29], it means that 10–20% of the parasite individuals in the subpopulation are outcrossing.

The frequency of outcrossing is much less than that demonstrated

in another taeniid tapeworm Echinococcus granulosus, which were estimated as 74% [30]. Such a contrast is consistent with

extraordinarily large infrapopulations typical of E. granulosus in canid definitive hosts and thus the chance of mating is simply

higher than that of T. solium. Nevertheless, the estimated rate of outcrossing for T. solium was unexpectedly high when considering that these tapeworms are nearly always found in single-worm

infections in humans. However, we experienced a case of taeniasis

involving 20 T. solium adults in China [31], and we assume that the multiple infection of T. solium tapeworms is not so rare in endemic areas. Our result suggests that the chance of outcrossing

has been underestimated and establishes hybridization as a

common outcome for the Asian and Afro-American genotypes

in zones of contact or sympatry. Further epidemiological study on

taeniasis in Madagascar may contribute to a better understanding

of the breeding systems of T. solium.

Conclusions

In the present study, we show that T. solium was introduced and established on Madagascar at least twice in the past 2000 years. An

Asian origin, from the Indian Subcontinent, for some genotypes of

T. solium contrasts with the established history and ancestry of the Malagasy culture primarily from ISEA. Our results demonstrate

that tapeworms from geographically disjunct regions in Africa or

Latin America and the Indian Subcontinent are now in secondary

contact on Madagascar following a history of isolation for

populations that may extend to the Pleistocene. Parasites with

origins in Africa/Latin America or Asia reflect the complex history

of development of the Malagasy culture, and in this case provide

compelling evidence for the history of human occupation of the

island. Our study highlights the importance of elucidating the

determinants for distributions of human pathogens and is

especially relevant given manifestation of distinct disease syn-

dromes and socioeconomic impact associated with the two

recognized genotypes of T. solium [4,32].

Supporting Information

Table S1 Location of the slaughterhouses and the numbers of pigs and cysts examined in each location.

(DOC)

Table S2 Nucleotide substitutions of mitochondrial cob gene in 22 haplotypes of T. solium.

(DOC)

Table S3 Nucleotide substitutions of mitochondrial cox1 gene in 23 haplotypes of T. solium.

(DOC)

Table S4 PCR primer pairs used for the amplification of nuclear gene markers.

(DOC)

Acknowledgments

The authors are grateful to Ms. Toshiko Miura and Tomoe Nakayama for

their kind support in molecular analyses.

Author Contributions

Conceived and designed the experiments: AI JC MN. Performed the

experiments: TY. Analyzed the data: TY. Contributed reagents/materials/

analysis tools: TY. Wrote the paper: TY JC YS MN EH AI.

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