Parasitology International 57 (2008) 256–270

Contents lists available at ScienceDirect

Parasitology International

j ourna l homepage: www.e lsev ie r.com/ locate /par in t

The distribution of Mekong schistosomiasis, past and future: Preliminary indicationsfrom an analysis of genetic variation in the intermediate host

Stephen W. Attwood a,b,⁎, Farrah A. Fatih b, Ian Campbell c, E. Suchart Upatham d

a State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, PR Chinab Department of Zoology, The Natural History Museum, Cromwell Road, London SW7 5BD, United Kingdomc Environment Department, Ok Tedi Mining Limited, P.O. Box 1, Tabubil WP, Papua New Guinead Department of Medical Science, Faculty of Science, Burapha University, Bangsaen, Chonburi 20131, Thailand

⁎ Corresponding author. State Key Laboratory of Bioth4 Lu, Chengdu 610041, PR China. Tel.: +86 28 8516 4098

E-mail address: swahuaxi@yahoo.com (S.W. Attwoo

1383-5769/$ – see front matter © 2008 Elsevier Irelanddoi:10.1016/j.parint.2008.04.003

A B S T R A C T

A R T I C L E I N F O

Available online 8 April 2008

Neotricula aperta is the on

Keywords:Neotricula apertaSchistosoma mekongiCambodiaLao PDRPopulation genetics

ly known intermediate host of Schistosoma mekongi which infects humans inCambodia and the southern tip of Lao PDR. DNA-sequence data (partial rrnL, i.e., mitochondrial 16S largeribosomal-RNA gene) were obtained for 359 N. aperta snails sampled at 31 localities in Cambodia, Lao PDRand Thailand. A nested clade analysis was performed to detect and evaluate any geographical patterns in theobserved variation and to identify genetic subpopulations or clades. Coalescent simulations were used tocompare different historical biogeographical hypotheses for N. aperta and S. mekongi. A coalescent basedmethod was also used to provide maximum likelihood estimates (MLEs) for effective populations sizes andhistorical growth and migration rates. Dates were also estimated for phylogenetic events on the gene treereconstructed for the sampled haplotypes (e.g. the time to most recent common ancestor). N. aperta wasfound to be divided into two monophyletic clades, a spring-dwelling form of northern Lao PDR and a morewidespread larger-river dwelling form of southern Lao PDR and Cambodia; this divergence was dated at9.3 Ma. The populations with the largest estimated population sizes were found in the Mekong River of LaoPDR and Cambodia; these, together with those of the rivers of eastern Cambodia, appeared to have been thefastest growing populations. Dominant levels of gene-flow (migration) were apparent in a South to Northdirection, particularly out of seeder populations in the Cambodian Mekong River. The radiation of N. apertainto sub-clades across Cambodia and Lao PDR is dated at around 5 Ma. The findings suggest that historicalevents, rather than ecology, might best explain the absence of S. mekongi from most of Lao PDR. The publichealth implications of these findings are discussed, as are pointers for future studies and surveillance.

© 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Mekong schistosomiasis has posed a persistent public healthproblem since its discovery in 1957 [1–6]. The causative agent ofMekong schistosomiasis is the parasitic blood fluke Schistosomamekongi Voge, Buckner and Bruce, 1978 (Trematoda: Digenea). Theintermediate host is the caenogastropod snail Neotricula aperta(Temcharoen, 1971) (Gastropoda: Pomatiopsidae: Triculinae). Threestrains of N. aperta have been identified on the basis of shell size andbody pigmentation [7]. All three strains are, to varying degrees(γ≫β≫α), capable of acting as host for S. mekongi, but only the γ-strain is known to be epidemiologically significant [8]. The first masstreatment programme for Mekong schistosomiasis began in 1989 atKhong Island in southern Lao PDR (Fig. 1); this was WHO coordinatedand involved praziquantel administration to riparian communities [9].

erapy, Gaopeng Street, Keyuan; fax: +86 28 8516 4092.d).

Ltd. All rights reserved.

In spite of 9 years of control efforts in Cambodia and Lao PDR, theprevalence in Hat-Xai-Khoun village, Khong Island, is still 26.8% (buthas decreased from almost 80% in 1989) [9] and at Sadao in Cambodia(Fig. 1) the prevalence rose from 0% in 2004 to around 2% in 2005 [6].The role of reservoir hosts in the persistence of transmission has beendemonstrated [10,11]. In addition, it has been shown that prevalence ofinfection in the snails at Khong Island [12] and Sadao [13] has notdeclined significantly in the face ofmarked reductions in prevalence inthe human population. Such observations suggest that control ofthis disease will be difficult to maintain and that the factors drivingreemergence of infection need to be understood.

In 1969 the estimated human population at risk from Mekongschistosomiasis was 150,000 and all known endemic foci were alongthe lower Mekong River in Cambodia and the southern tip of Lao PDR(i.e., over a distance of about 200 km). Prior to 1991 N. aperta wasthought to occur in the Mekong River only with a patchy distributionbetween Kratié and Stung-Treng in Cambodia, at Khong Island andaround the juncture of the Mul and Mekong rivers in Thailand (Fig. 1).The first report of N. aperta outside the Mekong and Mul rivers was

257S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

published in 1999 [14] and in 2004 details of 11 new populations,occurring in six river systems of Lao PDR and Cambodia, werepublished [13]. It was originally assumed that the absence of S.mekongi transmission from most of Lao PDR was due to ecologicalconditions unfavourable for N. aperta outside the Cambodian MekongRiver. The discovery of many N. aperta populations in the streams andsmall rivers draining the Annam highlands of Central Lao PDR raisestwo important questions. First, is the current range of S. mekongilimited by ecological conditions or by historical processes? Such aprocess could be a recent colonization of Central Lao PDR by N. apertawith a lag in the corresponding expansion of the range of S. mekongi,or that N. aperta is refugial in Central Lao PDR and the history is one ofpopulation decline and fragmentation (with gradual loss of foci ofinfected snails). Second, what are the migration rates betweenpotential seeder populations in the Annam area and the MekongRiver which receives its drainage waters? If migration rates were highsuch seed populations would play a significant role in the restorationof snail populations in the lower Mekong after snail controlprogrammes had ceased. In addition, the existence of N. aperta inthe small streams, pools and springs around which villages are oftenbased, presents a different disease control challenge than transmis-sion restricted to only a large river like the Mekong; the occupationalexposure patterns differ, the affected villages are more difficult topredict and spread over a larger area than previously thought. Thepotential human population at risk has risen from 150,000 to over1.5 million following the discovery of these new snail populations.

In this study the above questions are addressed using a molecularsystematic and statistical biogeographical approach. The aims were touse nested clade analysis (NCA) to identify historical processes (such aspast range expansion or decline) and also to use coalescent-based [15]approaches to estimate population sizes, past growth and migrationrates and to compare alternative population histories. Severalhypotheses for the origins of N. aperta have been proposed, such asDavis's mid-Miocene radiation model [16] or the North to Southradiation models of Attwood et al. [17] and Davis, [18], and a recentlyproposed Plio-Pleistocene South to North model [19]). Both NCA andcoalescent methods were used so that the indications from theseunrelated approaches could be compared. In 2005 sampleswere takenfrom 10 previously unknown N. aperta populations, DNA-sequencedata obtained, and the data combined with those for 21 previouslyknown but unsequenced populations. The present authors had alreadyobtained (between 1999 and 2003) DNA-sequence data for 10additional populations and the 2005 data were added to thispreexisting data set. The sequences used here are relatively shortbecause prior to 2003 sequencing was less routine than today and theobjectivewas to cover asmany individuals and populations as possiblein order to capture all variation present, nevertheless the data setincludes 104 polymorphic sites of which 63 (12.5% of all sites) arephylogentically informative. As this was the first population geneticstudy of N. aperta, it was intended that the findings would provideestimates of population sizes, growth, migration and divergence dates.Such estimates could then be used as prior information in futureanalyses based on independent data sets, and in the identification ofkey populations or processes for further surveillance or study (e.g.,sources of colonists, polarity of migration). Consequently, in thepresent study, prior assumptions are kept to a minimum and theanalyses were performed in a step-wise manner, with the results ofeach step being used to guide the choice of input parameters forsubsequent steps.

2. Materials and methods

2.1. Sampling and DNA-sequencing

Samples were taken in Cambodia, Lao PDR and Thailand. Table 1gives details of sampling sites, dates of collection, strain of N. aperta

and sample codes. Snails were brushed off stones, washed, excesswater removed from shell and then fixed in 3 changes of 100% ethanol.Sub-samples were fixed in 10% neutral formalin; these specimenswere used to confirm identity of the taxa. Identification of snailsfollowed published accounts [7,20]. 21 of the 31 populations sampledwere previously unknown and/or unsampled, further details on 5 ofthe new populations are published elsewhere [13]. Procedures andPCR primers used for DNA extraction and sequencing are standard andare described in the literature [17]. The loci sampled were partialsequences of the cytochrome oxidase subunit I gene (cox1) and thelarge ribosomal-RNA gene (rrnL), both on the mitochondrial genome.Two mitochondrial genes were selected because, with their maternalpattern of inheritance, and smaller effective population size, theywere considered to represent potentially better recorders of phyloge-netic events at the intra-specific level. In addition, the loci targetedwere those within regions previously shown to exhibit ideal levels ofvariation in Schistosoma for this type of study [17], and for whichefficient and reliable primers were available for the rapid sequencingof samples from a large number of snails. The choice of loci alsoallowed extension of an existing data set for some of the populations.DNA extracts were not pooled and one DNA sequence thusrepresented one snail. Sequences were assembled and aligned usingSequencher (version 3.1 Gene Codes Corp. Ann Arbor, Michingan).DNA sequences for both strands were aligned and compared to verifyaccuracy. Controls without DNA template were included in all PCRruns to exclude any cross-over contamination.

2.2. Phylogeny reconstruction

The datawere grouped into sets of aligned DNA-sequences of equallength (one for each locus) such that all individualswere represented ineach set. Theβ-strainwas included in thenested clade analysis to checkthat it formed a cohesive clade distinct from the γ-strain, but it wasomitted from the maximum likelihood analyses in order to reduce runtimes (which in some cases were up to 4 days) and demands oncomputermemory. The datawere analysed using a Bayesianmethod asimplemented by the software suite P4 [21]. A suitable substitutionmodel was selected using an hierarchical test of alternative models bymixed χ2-test as performed by Modeltest v.3.06 [22]. The modelparameters were then ‘optimized’ using a maximum likelihoodmethod (ML) with the Brent Powell algorithm in P4. The values fromthese optimizations were used as starting parameters for the firstBayesian analysis, as well as to define a model of substitution for thecoalescent simulation of DNA sequences when testing phylogeograh-phical hypotheses. Importance sampling with a Metropolis-coupledMarkov chainMonte Carlo sampling process (McMcMC) [23]was used.The Bayesian method in P4 was used to estimate initial gene andpopulation trees because of its speed and the ability to afford thesimultaneous estimation of topology and confidence levels (i.e., theposterior probabilities) for the nodes inferred. The first 25% of samplesfrom the posterior distribution was discarded as a ‘burnin’. Probabil-itieswere then estimated over 900,000 generations beyond the burnin.Four simultaneous Markov chains were run (one cold, three heated)and trees were sampled every 10 generations, two such runs wereperformed simultaneously. After 900,000 generations (post-stationar-ity) the average standard deviation of the split frequencies (betweenthe 2 runs) was checked; theMcMCwas considered complete if this SDwas b0.01.

2.3. Population genetics

Pairwise mismatch distributions calculated in Arlequin [24,25] forthe clades identified by NCA were used in the initial inspection of thedata for evidence of population expansion; those populations whichhave undergone recent expansion are expected to show a unimodaldistribution, whereas those with a history of relatively constant size

258 S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

Table 1Locations and dates of the Neotricula aperta collections

River Territory Collection code Province Village Location (GPS) Collection date Infection N

Mekong Cambodia KRK Kratié Krakor 12°30′15″; 106°01′00″ 07/05/01 No 18Mekong Cambodia KSC Sambour San Dan 12°44′20″; 106°00′30″ 10/05/01 Yes 9Mekong Cambodia SST Stung-Treng – 13°24′00″; 105°56′00″ 29/04/03 No 15Mekong Cambodia XKS Sambok Xai-Khoun 12°37′15″; 106°01′15″ 09/05/01 Yes 6Mul Thailand BET Ubon Kaeng-Kao 15°14′00″; 105°16′00″ 06/04/05 No 12Mekong Laos DKL Champassac Don Khong 14°06′30″; 105°51′15″ 03/04/01 Yes 19Mekong Laos DNL Champassac Don Nang Loi 14°24′30″; 105°05′45″ 03/04/01 No 9Mekong Laos DPK Champassac Don Phakan 13°56′00″; 105°56′30″ 03/04/01 No 8Mekong Laos HXK Champassac Hat-Xai-Khoun 14°06′00″; 105°52′00″ 03/04/01 Yes 11Mekong Laos MOP Champassac Mophou 14°59′15″; 105°54′00″ 03/04/01 No 16Mekong Laos NFL Champassac Na Fang 14°28′00″; 105°52′00″ 03/04/01 No 9Mekong Thailand DAN Ubon Dan 15°19′00″; 105°31′00″ 13/05/02 No 8Mekong Thailand BKL Ubon Khi-Lek 16°02′15″; 105°18′00″ 07/04/01 No 8Sre Pok Cambodia OHG Rattanakiri Oh Gan 13°29′30″; 106°52′30″ 26/04/04 No 11Sre Pok Cambodia DIL Rattanakiri Diloh 13°28′45″; 107°00′15″ 25/04/04 No 14Sre Pok Cambodia TAL Rattanakiri Jua Talai 13°28′30″; 106°59′45″ 25/04/04 Yes 17Sre Pok Cambodia JND Rattanakiri Jua Negn Dai 13°27′30″; 106°52′45″ 26/04/04 No 19Xe-Bang-Fai Laos MXL Khamouane Mahaxai 17°25′00″; 105°11′45″ 20/04/01 No 10Xe Don Laos BHD Saravane Houay-Deng 14°44′45″; 106°38′15″ 04/04/03 No 10Xe Don Laos XDN Champassac Hom-Sak 15°42′30″; 106°04′15″ 03/04/03 No 9Xe Kaman Laos XKM Attapeu Kong-Phine 14°46′15″; 106°50′15″ 08/05/03 No 11Xe Kong Cambodia KRN Stung-Treng Karach Nun 13°45′00″; 106°12′15″ 25/04/03 No 10Xe Kong Cambodia SDO Stung-Treng Sadao 13°36′45″; 106°06′00″ 25/04/03 Yes 10Xe Kong Laos XKG Attapeu Attapeu 14°48′30″; 106°49′00″ 08/05/03 No 10Xe kong Laos DKX Attapeu D. Kaeng Xai 14°45′45″; 106°45′00″ 08/05/03 No 8Nam Hinboun Laos KLR Savanakhet Kong Lor 17°57′30″; 104°44′00″ 26/03/04 No 17Nam Yom Laos YOM Khamouane Yommarat 17°36′15″; 105°10′30″ 20/04/01 No 7Nam Yom Laos TOT Khamouane Thathot 17°37′30″; 105°08′45″ 28/03/04 No 16Nam Pakan Laos TKN Khamouane Thakhen 17°41′15″; 104°41′45″ 27/03/04 No 15Xe San Cambodia RAM Stung-Treng Krabi Chum 13°33′15″; 106°31′00″ 24/04/03 No 14Xe San Cambodia SRG Stung-Treng Sri Goh 13°36′30″; 106°22′15″ 24/04/03 Yes 10

Populations for which DNA-sequence data have not been previously published are given in bold (see Collection code). For spring or primary-stream populations the river fromwhich they were collected (or, in cases where the stream was too small to be named, the major drainage with which they are associated) is given in italics. The number ofindividuals sequenced (N) and whether any infection with S. mekongi was detected is also given for each locality. Collection date is dd/mm/yy. A — is inserted where there is nonearby village.

259S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

have an expected multimodal distribution [26]. Harpending's ragged-ness index [27] was used to assess the significance of any deviationfrom the pattern expected under the null hypothesis of constantpopulation size. Genetic diversity of haplotypes was also exploredinitially using AMOVA (and the K2P distance in Arlequin) to assess theextent to which genetic variation was partitioned among clades iden-tified by the genealogical analyses. A total of 1000 randomizationswere used to assess significance of variance estimates for the ϕ-statistics [24]. Population genetic parameters such as uncorrectedaverage pairwise sequence divergences (dxy), nucleotide diversity (π)[28], number of polymorphic sites, Tajima's D [29], etc., wereestimated using DNAsp [30]. A molecular clock calibration was per-formed for these data following the method of Edwards and Beerli[31]. DNAsp was used to obtain dxy values between corresponding 16Ssequences for Robertsiella silvicola and N. aperta from the GenBank(accession numbers AF531548 and AF531556, respectively [17]. Thetwo snail taxa were assumed to have diverged 2.1 Ma; this was on thebasis of divergence times for S. malayensis and S. mekongi (the res-pective Schistosoma species hosted by the snail taxa), estimatedelsewhere using a Bayesianmethod applied to parasite DNA-sequencedata and the timing of Pliocene fluctuations in sea level [19,32]. Theabove procedure yielded a simple point estimate for the mutation rateat the rrnL locus of 1.095±0.17×10−8 substitutions per site per year.The current understanding of triculine phylogeography is insufficientto permit a more rigorous estimation procedure than this.

Fig.1. Sketchmap of the Annam and Kontum region of Southeast Asia showing the locations ofoci for Schistosoma mekongi (denoted by black box symbols). Important geographical featurmain text and tables, is the lower Mekong river between Paxse in Laos and the Cambodian

2.4. Phylogeographical analyses

The statistical parsimony procedure [33] implemented in theprogram TCS v.1.18 [34] was used to estimate an unrooted haplotypenetwork for the rrnL and cox1 data sets separately. Assessment of thegenealogy in the form of a networkwas consideredmore realistic thanthe bifurcating trees handled by phylogenetic methods (as in P4 or MLin PAUP⁎ [35]) because of the simultaneous occurrence of ancestraland descendant forms in a gene tree (in species phylogenies ancestralforms (the nodes) will be extinct). Statistical parsimony is advanta-geous in that it emphasizes the similarities (rather than differences)among haplotypes, and is therefore well suited to reconstruction ofintraspecific relationships where levels of variation are expected to belower than at the species level [36]. The TCS algorithm [33] is fast evenwhen handling large data arrays; however the reliability of its treesearch suffers in that it is able to make only local (neighbour toneighbour) not global (i.e., across the whole tree) comparisons.Nevertheless, TCS allows us to estimate a set of 95% credible networksin that haplotypes separated by ≤9 mutational steps had a ≥95%chance of being connected in a parsimonious manner. Haplotypeswere nested according to the conventional rules [37]. Briefly, thenesting procedure begins at the tips of the network and, moving onemutational step inwards, haplotypes are grouped into 1-order clades.The process is repeated, nesting the 1-order clades into 2-order clades,et seq. until all subclades have been nested into a single clade; this was

f theNeotricula aperta populations sampled (denoted by star symbols) and transmissiones or locations are given in large bold type. The “Champassac region”, referred to in theborder.

Fig. 2. Population trees representing different kinds of historical biogeographical hypotheses explaining the current distribution of Neotricula aperta across Laos and Cambodia.Branch widths are proportional to the estimated effective population size. A. A single historical divergence event into two effectively panmictic populations, a spring dwelling and ariver dwelling form of N. aperta. B. An original 502 bp haplotype gives rise to a 503 bp haplotype, with a subsequent divergence to produce a third lineage bearing a 504 bp haplotype.C. A ‘star-tree’ model, with all populations diverging simultaneously from a single ancestral population or arising from fragmentation and decline of a once larger and continuouspopulation. D. An example of a more complex history (in this case of a northern/Attapeu original population with subsequent North to South radiation).

260 S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

the basis of the NCA. Ambiguities were resolved following Crandallet al. [38]. GeoDis v.2.2 [39] was used to test for any significantassociation among sampled haplotypes and the spatial distribution of

Fig. 3. Haplotype network showing the higher order clades from the nested clade analysishaplotypes (in the total sample). The broken line between clades 4-3 and 4-5 represents an abroken in several places. All haplotypes unique to spring dwelling taxa are encompassed by

the individuals bearing them. Geographical distances betweensampling sites were measured both as ‘along-river’ distances (alongcontiguous or nearly contiguous stretches of river systems separating

for Neotricula aperta. Clade diameter is proportional to the frequency of the containedmbiguous connection, i.e., the haplotypes are connected by a closed loop which could beclade 5-3.

Table 2Basic descriptive statistics and geographical distribution for each higher clade in the NCA

Clade Haps N sites N indiv Nuc div K S T Geographical representation

4-1 18 503 47 0.0026 1.31 17 b0.01 CER (0) CLM (100) LAR (0) LMK (0) NSP (0)4-2 8 503 80 0.0054 2.71 30 N0.05 CER (100) CLM (0) LAR (0) LMK (0) NSP (0)4-3 30 503 37 0.0119 5.99 27 N0.05 CER (30) CLM (20) LAR (40) LMK (10) NSP (0)4-4 46 503 41 0.0143 7.21 29 N0.05 CER (0) CLM (0) LAR (43) LMK (57) NSP (0)4-5 13 503 33 0.0097 4.89 25 N0.05 CER (25) CLM (0) LAR (13) LMK (62) NSP (0)4-6 18 503 46 0.0035 1.78 27 b0.001 CER (0) CLM (0) LAR (0) LMK (100) NSP (0)4-7 15 502 39 0.0176 8.83 32 N0.05 CER (0) CLM (0) LAR (25) LMK (63) NSP (12)4-8 10 504 17 0.0071 3.56 11 N0.05 CER (0) CLM (0) LAR (0) LMK (0) NSP (100)4-9 4 503 19 0.0058 2.93 17 N0.05 CER (0) CLM (0) LAR (0) LMK (0) NSP (100)5-1 94 503 125 0.0140 7.06 50 N0.05 CER (18) CLM (24) LAR (35) LMK (23) NSP (0)5-2 39 503 159 0.0093 4.66 66 b0.05 CER (47) CLM (0) LAR (6) LMK (47) NSP (0)5-3 29 502 75 0.0218 10.9 46 N0.05 CER (0) CLM (0) LAR (30) LMK (40) NSP (30)

Haps, number of haplotypes; N sites, number of sites analyzed (excluding gaps); N indiv, number of individuals in clade; Nuc div, Jukes-Cantor corrected nucleotide diversity πJC; K,average number of pairwise nucleotide differences; S, number of polymorphic sites; T, significance of Tajima's test for neutrality (significant tests in bold). Geographicalrepresentation is a percentage based on the number of villages from each region represented in the clade: codes; CER, eastern rivers of Cambodia (Sre Pok, Xe San and lower Xe Kong);CLM, Cambodian section of the lower Mekong river; LAR, southern Laos — Attapeu region (upper Xe Kong and Xe Don, etc., not Mekong river); LMK, Laotian section of the lowerMekong river; NSP, northern Laos (not Mekong) including all of the spring and very small river populations. Clades with haplotypes carried by members of populations known to beinfected by Schistosoma mekongi are given in bold.

261S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

them) and as direct, shortest, distances calculated using the GPS.GeoDis computes the following 4 statistics together with theprobability and the direction of their deviations from null expecta-tions: clade distance, Dc (a measure of the geographical range of aparticular clade), nested clade distance Dn (a measure of thegeographical distribution of a clade relative to its nearest sisterclade; these are used to derive the average interior clade distanceminus the average tip distance ((I−T)c and (I−T)n, respectively).

2.5. Coalescent based analyses of demographic history and levels ofmigration

A coalescent approach, as implemented in Fluctuate [40], was usedto estimate the past exponential growth rates for each major cladefrom the NCA. The estimation procedure used 20 short chains of 1000generations and 5 long chains of 20,000 generations. Starting valuesfor each run were initial g (exponential growth rate) 0.001,Watterson's 1975 estimate of Θ, empirical nucleotide frequencies,transition/transversion ratio 20.47 (from P4), and a UPGMA startingtree (HKY85 distance) for the sequences (from PAUP⁎). The procedurewas run 5 to 8 times until consistent estimates were returned. Thestarting parameters of subsequent runs were the maximum likelihoodestimates (MLEs) from previous runs.

A coalescent based approach was also used to obtain MLEs formigration (or gene-flow) rates among the higher clades identified byNCA. The procedure used was that in MIGRATE v.0.97 [41]; unlikeequilibrium approaches this model is able to incorporate populationstructure and processes such as past asymmetrical gene-flow. Theprocedure assumes the infinitive alleles model. Typical runs used 20short-chains (of the McMcMC) with 100 steps and 2000 recordedgenealogies, and 4 long chains with 20 steps and 10,000 recordedgenealogies (500,000 for 5-order clades). AIC-modelTest was set tofast, likelihood estimation to slow, adaptive heating was used andchains were combined over 5 multiple runs. For the first runs startingvalues for 2 Nm were estimated separately (by specifying migrationmodel matrices with all other parameters fixed) prior to runs usingthe full migration model. The analyses were repeated until constantvalues were obtained (this required several weeks of computing timeon a Windows XP PC, 1.4 GHz processor, 256 MB RAM).

2.6. Summary statistics for hypothesis testing regarding populationhistory and timing of phylogenetic events

Hypothesis testing followed a method described in Knowles andMaddison [42], which (unlike NCA) permits the building of specific

distributions for the data and plausible histories, thereby enablingstatistical tests of alternative phylogeographies. Briefly, data setssimulated by a neutral coalescent process were used to estimate thedistribution of the chosen test-statistic under the population history(the hypothesis) being tested. The value of the test-statistic computedfor the real data set is compared to this distribution and itssignificance determined. The test-statistic used was Slatkin andMaddison's [43] s statistic, which measures the discord between thegenetree for the data and the subdivisions of populations on thepopulation tree (which represents the historical-biogeographicalhypothesis under test). The s statistic regards the subpopulations ascategorical variables and in this case can be regarded as the minimumnumber of migration events, among populations, required to explainthe population tree in terms of the gene tree (s is strictly a measure ofcompleteness of lineage sorting). Unlike NCA this process is too slowto examine all plausible population histories and cannot test multiplehistories simultaneously. Consequently, a selective decision must bemade about which hypotheses to test. MESQUITE v.1.05 [44] was usedto perform the coalescent simulations; the hypotheses tested aredetailed in Fig. 2. In order to compared simulated (as evolving underthe history of interest), reconstructed, gene trees with the empirical,reconstructed, gene tree, 1000 DNA-sequence data sets were simu-lated (using the model parameters from P4) and 1000 correspondinggene trees were estimated using PAUP⁎ (heuristic search with theparsimony criterion).

Finally the program Genetree [45] was used to estimate the ages ofmutations defining clades (i.e., the clades identified by earlieranalyses). Genetree implements a likelihood based method usingimportance sampling and allowed the incorporation of growth andmigration rates (usingMLEs from the earlier analyses as initial values).All sites that were incompatible with the infinite sites model wereremoved prior to the analysis. Ancestral states were determined usingcorresponding sequence data for Tricula bollingi and Neotricula burchifrom the GenBank (accession numbers AF531551 and AF531542). Eachrun of Genetree produces a possible ordering of coalescent andmutation events explaining the data. The distribution of the timesbetween these events is known; therefore the ages of key mutationevents and the TMRCA can be estimated. The simulated ages areweighted by the probability of that run and the MLEs were theweighted averages of the simulated ages over up to 10,000,000 runs.The runs were conditioned on estimates of the effective populationsize (Ne) from MIGRATE. The initial θ value was obtained from thenumber of segregating sites using DNAsp. Initially shorter chains wereused (≤30,000 runs) to estimate the back migration rate matrix andthen to find the likelihood for different pairs of θ and β (the

262 S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

Table 3Inferences from the NCA (see Templeton [58] for details)

Clade χ2-statistic Probability Inference chain Inferred demographic event

R3-1 14.262 0.001 1-2-11-17 No InconclusiveR3-5⁎ 29.000 b0.0001 1-2-3-4 No RGF with IBD in the Champassac, upper Xe Kong and Attapeu regionsR3-8 15.273 0.05 1-2 InconclusiveR3-15 15.000 0.003 1-19-2-11-12 No CRE in the Champassac, Lao Mekong river and Attapeu regionsR4-2 72.095 b0.0001 1-2 InconclusiveR4-3 27.076 b0.0001 1-2-3-4 No RGF with IBD in upper reaches of Xe Kong and eastern Cambodian riversR4-4 53.486 b0.0001 1-2-11-12 CRE in the Laotian lower Mekong/Attapeu regionR4-5 66.00 b0.0001 1-2-3-4 No RGF with IBD in upper Xe Kong and Champassac Mekong river regionG3-1 14.262 0.001 1-2-3-4-9 No AF in Cambodian lower Mekong regionG3-5a 29.000 b0.0001 1-19-20-2-3-4-9 No AF in Champassac, upper Xe Kong/Attapeu regionG3-8 15.273 0.05 1-2 InconclusiveG3-15 15.000 0.003 1-19-2-11-12 No CRE in the Champassac, Lao Mekong river and Attapeu regionsG4-2 72.095 b0.0001 1-2 InconclusiveG4-3 27.076 b0.0001 1-2-3-4-9 No AF in upper reaches of Xe Kong and eastern Cambodian riversG4-4 53.486 b0.0001 2-3-5-6-7-8 Yes Probably past gene-flow in LMK & LAR regions, then extinction of intermediate populationsG4-5 66.00 b0.0001 1-19-20 No Inadequate geographical sampling (otherwise CRE would be inferred)

Clades without significant geographical associations and 1-order and 2-order clades are not listed. An R prefix to the clade name signifies that distances were measured alongconnecting rivers, whereas a G prefix indicates that shortest (direct) distances between localities were used. AF, allopatric fragmentation; CRE, contiguous range expansion; IBD,isolation by distance; RGF, restricted gene-flow.

a Contained the β-strain samples.

263S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

exponential growth rate parameter), alternately fixing θ and varyingβ, or vice versa; this required about 30 sets of runs. A migration ratematrix was then re-estimated with θ and β fixed to their MLEs (ornear to their MLEs). A likelihood ratio test was used to compare theresults with a migration matrix and without migration. Finally thegene tree and MLEs for the ages of nodes were computed using these‘optimal’ starting parameter values and 10,000,000 runs, with 1000points sampled from the empirical distribution and an expectedmaximum for the TMRCA of 18 Ma (the probable date for the arrival oftriculine snails in China from their origin in India [18]). Probabilityintervals for the TMRCAwere obtained from the approximate area of aplane that follows the TMRCA on the likelihood surface. Dates inmillions of years before present (Ma) were estimated from coalescent‘time’ units using a generation time of 0.25 years for N. aperta. Thepresent generation time of Mekong River N. aperta is not knownprecisely [46], but most triculine snails in tropical areas achieve 4generations per year and it is assumed that spring and streamdwelling (antecedent) N. aperta populations achieved this reproduc-tive rate during most of the species' history.

3. Results

3.1. Nested clade analysis

DNA sequencing provided 598 bp at the cox1 locus and 502–504 bp at the rrnL locus. The cox1 data appeared saturated accordingto the test of Xia et al. [47], showed many incompatibilities with theinfinite sites model and the cox1 haplotypes could not be united intoan unambiguous network by statistical parsimony. Consequently, thecox1 data were not included in the statistical analyses. For the rrnLlocus sequences, were obtained for 359 snails representing 31populations, 162 haplotypes, 10 river systems, 11 provinces or districtsand 3 countries. These sequences were deposited in GenBankwith accession numbers EU306175–EU306335. The nesting processresulted in 3 5-order clades, 9 4-order clades, 22 3-order clades, and42 2-order clades. AMOVA, with haplotypes partitioned according to5-order clades (effectively corresponding to major river systems), 4-order clades (sub-populations within river systems) and within 4-order clades, suggested that most of the variance (89.27%) occurred

Fig. 4. A haplotype tree estimated using a Bayesian method in P4. The tip of each branch isparentheses. Solid branch lines indicate haplotypes 502 bp in length, lines with short dashposterior probability of the clade to the right.

between the 4-order clades (Pb0.001). Consequently, and as thenumber of haplotypes and subclades was large, only 4- and 5-orderclades are shown in Fig. 3 (the summary of the haplotype network)and in Table 2 (details of geographical extent and population geneticparameters by clade). The nested cladogram provided a moresimplified depiction of the relationships among haplotypes than didthe Bayesian gene tree (Fig. 4). Table 2 shows that there was nophylogenetic constraint to compatibility with S. mekongi as 6 out ofthe 9 4-order clades and all 3 5-order clades were involved intransmission. Clades 4-1, 4-6, 4-8 and 4-9 were geographicallyhomogeneous, being found only in regions CLM, LMK, NSP and NSPrespectively (see Table 2 for codes). The clades with the highestnucleotide diversities (4-7, 4-4 and 4-3) were comprised of 50 to 100%of either the LAR or LMK populations andwere the clades at the centreof the modern range of N. aperta. It is reasonable to expect thosesubpopulations (as identified by NCA, sequence length variation orother reasoning) that show greatest internal genetic diversity to beancestral. The nucleotide diversities for clades 4-7, 4-4, 4-3 and 4-5were significantly greater than that of the next most diverse clade 4-8(Pb0.05; based on a distribution of nucleotide diversities for a set of 20haplotypes simulated usingMESQUITE and parameters taken from theingroup (empirical) data). CLM and NSP were the most phylogeneti-cally restricted regions (represented in only 2 and 3 4-order clades,respectively), whilst LMK, LAR and CER were the least restricted. Theclades with the most geographically widespread haplotypes were 4-3,4-5 and 4-7. All of the NSP samples fell into clades 4-7, 4-8 and 4-9(clade 4-8 comprises solely of KLR haplotypes) and all of the CLMhaplotypeswere in 4-1 and 4-3. All of the Cambodian haplotypeswereencompassed by clades 4-1, 4-2 and 4-5. Clades 4-1 and 4-2 areentirely Cambodian, and clades 4-4 and 4-6 to 4-9 are entirely Laotian.Clades 4-1 and 4-6 show significant values for Tajima's D; this is a testfor the absence of selection, however, in the present case it is alsopossible that the test had detected marked population expansionrather than selection [48]. The null hypothesis of no associationbetween genetic variability and geographical distribution could berejected for 25 1-order, 10 2-order, 4 3-order and 4 4-order clades.Table 3 shows the inferences for from the NCA for 3- and 4-orderclades with significant χ2-values only. The inferences varied depend-ing on the distancemeasure used. For the river distance based analysis

labelled with the haplotype and the clades (from the nested clade analysis) involved ines 503 bp and lines with long dashes 504 bp. Numbers next to each note indicate the

Table 4Gene-flow estimates (2 Nm) among the 4-order clades of the NCA

4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9

4-1 – 0.0721 1.7931 b0.0000 0.0000 b0.0000 b0.0000 0.0316 b0.00004-2 0.1925 – b0.0000 1.2733 0.0142 0.1637 b0.0000 b0.0000 b0.00004-3 0.1418 0.0720 – 0.2315 0.1987 b0.0000 b0.0000 b0.0000 b0.00004-4 b0.0000 0.0481 0.1216 – 0.0000 b0.0000 b0.0000 b0.0000 b0.00004-5 b0.0000 0.1154 0.0608 b0.0000 – 0.0455 b0.0000 b0.0000 b0.00004-6 b0.0000 b0.0000 0.0304 b0.0000 0.2981 – 0.3429 b0.0000 b0.00004-7 0.0172 b0.0000 b0.0000 b0.0000 0.0000 0.0273 – 0.0316 b0.00004-8 b0.0000 b0.0000 b0.0000 b0.0000 0.0000 b0.0000 0.6857 – b0.00004-9 b0.0000 b0.0000 b0.0000 b0.0000 0.0000 b0.0000 b0.0000 b0.0000 –

Recipient populations are arranged by columns, donor populations by rows (likelihood of the model − ln(L) 232.097). For ease of interpretation flow rates above 0.1 are marked inbold.

Table 5Gene-flow estimates (2 Nm) among the 5-order clades of the NCA

5-1 5-2 5-3

5-1 – 1.2889 0.01005-2 1.1143 – b0.00005-3 b0.0000 b0.0000 –

Recipient populations are arranged by columns, donor populations by rows (likelihoodof the model − ln(L) 3214.3449).

264 S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

the key trend was restricted gene-flow (with isolation by distance) inthe Champassac area, the eastern Cambodian rivers and in particularthe upper Xe Kong River; this scenario implies that the geographicaldistribution of a haplotype will be strongly correlated with its age. Aweaker trend was contiguous range expansion in the lower Mekongbelow Champassac and the area around Attapeu (under this scenarioolder haplotypes will be restricted to the pre-expansion area). WhenGPS distance measures were used the result for clade 3-15 wasunchanged but the inferences of restricted gene-flow were replacedby allopatric fragmentation for the LAR and LMK region (clade 4-4showed past gene-flow with extinction of intermediate populations)rather than contiguous range expansion. The inconclusive results inTable 3 are due to an absence of tips in the clade concerned; theinadequate geographical sampling of clade 4-5 results from adoptionof a conservative assumption that unsampled N. aperta populationsdid occur between the collecting sites (contiguous range expansionwould have been the inferred process had all populations beenassumed to have been sampled). The by-river-distance distancemeasure is considered to be more realistic than the GPS distance, asthe latter does not allow for highland barriers or gradient.

3.2. Maximum likelihood analysis of demographic history

Tables 4 and 5 give the gene-flow estimates among the cladesidentified by NCA. Fig. 5 depicts the geographical extent of each 4-orderclade, whilst Fig. 6 shows the major tracts of gene-flow, areas ofpopulation expansion and of restricted gene-flow. The coalescent basedestimates of gene-flow show no contradiction with the results of theNCA in that area of restricted gene-flow. NCA (Fig. 6) also shows lowrates in Table 4. Table 4 suggests that gene-flow in a South to Northdirectionhas dominated the overall history ofN. aperta, the source beingclade4-1 in theCambodianMekongRiver, followedbyEast toWest fromthe Cambodian Sre Pok River into the Mekong River near Khong Island.The migration rates among the 5-order clades simply reflect those oftheir constituent 4-order clades. Themainmigration tract appears to befrom Cambodia to Lao PDR.

Values of the exponential growth rate parameter (g) for eachhigher order clade, estimated using Fluctuate, are given in Table 6.Very high growth rates were estimated across all Cambodian clades(ranging from 1584 to 4443) and also for the lower Mekong River(LMK) centered around Khong Island (2859). The lowest growth rateswere estimated for the lower Xe Kong, the Mekong River fromNortheast Thailand to Pakxé and the northern spring populations (−76to 108). Clade 4-8 (comprising solely of haplotypes from KLR) fromnear the Nam Theun II dam construction project was the only one ofthe NSP populations to show marked exponential growth (g=520).Clades 4-6 and 4-1 showed the highest estimated Ne values and 4-9

Fig. 5. The approximate geographical extent of the clades identified by the nested clpopulation size (as estimated by a coalescent method). The thick broken line along th

and 4-5 the lowest (Fig. 5). The raggedness index from the mismatchdistribution was significant for clades 4-9, 4-7, 4-5, 4-4 (Pb0.01) and4-8 (Pb0.05); this suggests that clades 4-1, 4-6, 4-2, and 4-3 areexpanding populations.

3.3. Statistical tests of population history

Coalescent simulations conducted in MESQUITE enabled rejectionof a ‘star-tree’ model (Fig. 2C) (sobserved 39 Pb0.01). The history ofnorthern/Attapeu original populationwith subsequent North to Southradiation (an hypothesis proposed in the literature on N. aperta [17])was also rejected (sobserved 39 Pb0.01). Indeed, all histories involvingthe 5 regional subpopulations were indistinguishable and rejected bythis procedure probably because of the complexity of the empiricalgene tree. Fig. 2C and D therefore represents all the more complexmodels. In contrast the histories in Fig. 2A and B could not be rejected(sobserved 7 and 22, respectively, PN0.05). A random distribution ofsimulated reconstructed gene trees indicated that the probability ofobserving this difference in s values by chance (under eitherpopulation history (A or B)) is b0.0001. Consequently, even accountingfor the fact that the simpler model would be expected to showa betterfit to the gene tree, the hypothesis of an ancestral population diverginginto spring and river forms, with the river form subsequently radiatinginto all the clades seen today, appears to be the more probable modelgiven the data (the gene tree). It was not possible to vary branchlengths and test different timings of events by this method becauselengthening the branches tends to decrease s anyway. Instead MLEsfor divergence times were obtained by other methods (see below).

3.4. Genealogy, phylogeography and dating

A gene tree for the sampled haplotypes was estimated using aBayesian phylogenetic method (in P4). There were 50 haplotypes(from the CER and LMK regions (mostly 504 and 505 bp in length) seeTable 7) whose relationships were entirely unresolved (Fig. 4). Severalparaphyletic clades were found (3 for CER and 3 for LMK) whoseinterrelationships could not be elucidated. Finally 3 monophyleticclades were observed, which encompassed all of the haplotypes in

ade analysis. The depth of shading of each clade is proportional to its effectivee Cambodian border approximately marks the uplifted edge of the Khorat Basin.

265S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

266 S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

their respective geographical regions; these were clades comprisingentirely of either CLM, or LAR or NSP haplotypes. The tree shows asecond lineage of NSP (sister to LMK 34-37), and a third with lower XeKong taxa and some LMK; however, these taxa, although in the NSPregion and having spring associated members, were in habitatscontiguous with true rivers and so were probably not true springdwelling forms (thus the main NSP clade is monophyletic). The genetree has 311 branches so posterior probabilities on the estimatedclades would not be expected to be high; nevertheless there wasreasonable support for the monophyletic clades (0.60, 0.72 and 0.91,respectively, as mentioned above) and the paraphyletic clades (0.61,0.64, 0.66, 0.89, 0.93, 0.94). The best supported clade was that for thetrue NSP (spring) populations.

There was no apparent phylogenetic constraint to compatibilitywith S. mekongi on the gene tree except that the true NSP clade had noassociated individuals from epidemiologically significant populations.All haplotypes from infected populations fell into either theunresolved haplotype ‘group’, the CER clade (CER40(CER11,CER12,CER13(CER14,CER15))) or the (LMK28,LMK14) clade. Haplotypes of all3 lengths were sampled from populations involved in S. mekongitransmission (16% 502 bp, 40% 503 bp and 44% 504 bp). The naturalinfection rates are low and the haplotypes of individual infected snailsare unknown; therefore it is impossible to discern which haplotypes(if any) are associated with infected individuals in a population. Table 8summarizes the results from the Genetree analysis. The findingssuggest an initial Miocene divergence of N. aperta into a springdwelling and a river dwelling lineage (10–9 Ma). The southernmost(CLM) populations appear oldest (around 10.12 Ma) and the radiationinto hilly areas (Annam) of Central Lao PDR is dated at around 6.5 Ma.Phylogenetic events also appear to have occurred within the NSP, CLMand LAR monophyletic clades in a burst between 4 and 6 Ma.

4. Discussion

4.1. Historical biogeography

The NCA (the results of which are summarized in Figs. 5 and 6)indicated restricted gene-flowwith isolation by distance in the East ofthe range (Attapeu, Kontum, Sre Pok, Xe San, upper Xe Kong region)and contiguous range expansion in theWest (along the Mekong River,down to Stung-Treng). The occurrence of isolation by distance in theEast is probably a Pleistocene–Holocene effect reflecting the hillynature of the Annam region with snails inhabiting streams isolated indifferent minor valleys. The range expansion inferred for the western(Mekong River) populations agrees with the estimated growth rates(Table 6). The MLEs for past growth rates were high for the MekongRiver (where clades 4-1 and 4-6 show the highest rates observed) andfor the eastern Cambodian rivers (with high rates of 36% and 21% ofthat of clade 4-1 observed for clades 4-2 and 4-3, respectively). Suchrates are in marked contrast to those of the predominantly northernLao (spring dwelling) and central Lao (Xe Bang-Fai, Xe Don and upperXe Kong) clades, which had growth rates only 1–4% of the MekongRiver and Cambodian clades. The only exception was clade 4-8, thespring dwelling population of Kong Lor which had a growth rate 12%of that of clade 4-1.

The above observations fit well with the idea of a spring dwellingancestral form of N. aperta giving rise to a more lentic ecotype, whichthen underwent marked range and population expansion in largerivers such as the Mekong [19]. Under such a hypothesis, it is likelythat the divergence of S.mekongi from an S. japonicum/S. sinensium-

Fig. 6. As Fig. 5 but key clades are unified by region andmajor tracts of gene-flow are shown bof the values for the unified clades). The inferences of the nested clade analysis are also showngene-flow. Historical processes inferred: an ancestral spring dwelling N. aperta clade in the Aand Cambodian Mekong rivers in independent migrations (6–5 Ma); after major populationregion (b5 Ma); spring ecotype is relict; Pleistocene uplifts break connections between Cam

like antecendent of Pliocene rodents occurred after the divergence ofthe river form of N. aperta. Such reasoning is based on the observationthat in primary streams rodents are more suitable hosts, whereashuman mediated transmission is more efficient in the shallow, clearwater, areas of the Mekong River where N. aperta occurs. The riverecotype of N. aperta is found in shallow areas characterized by sheetrock [49] and such areas are used by people for mooring boats,laundry, bathing children and other domestic activities. The concept oftwo ecotypes (a spring form and a large river form) appears fairly wellestablished in the present study. The highest levels of support on theBayesian gene tree are observed for the clade bearing the northernspring-dwelling forms. The distinction seems to be based on habitat,rather than geography (i.e., they are not simply united by beingnorthern populations) because those northern populations whichbordered or extended into larger rivers fell outside the true spring-associated clade (see Fig. 4). All of the haplotypes unique to springdwelling N. aperta occur in the same 5-order nested clade. Further, themost favoured population history in the coalescent simulations wasthat of a single divergence into a spring dwelling ecotype and a larger-river ecotype. The likelihood based method in Genetree gave a lateMiocene date for this original divergence of N. aperta into the twoecotypes; this date agrees with the general consensus of a mid-Miocene arrival of Triculinae in Yunnan, China [50].

A second population history, based on observed haplotype length(Fig. 2B), was also not rejected by the coalescent-simulation basedanalysis. This history (in approximate geographical terms) describesan initial divergence of a common ancestor into an Attapeu (Annam oreastern) clade and a Bolovens clade, with subsequent divergence ofthe Bolovens clade to produce a Kontum clade (see Table 7 for anexplanation of these terms, also see Fig. 1); this follows the “Red-riverhypothesis” [51] that N. aperta entered northern Lao PDR from Hunan(China), via northern Vietnam, and radiated in the Annam hills beforeinvading theMekong River. In further support of this model clades 4-7,4-3, 4-4 and 4-5 showed the greatest internal genetic diversity (π seeTable 2, Fig. 5) and these clades had the most widely dispersedhaplotypes. Clades exhibiting such properties are often referred to asgeographically ‘ancestral’ [52]; this would centre the origin ofN. aperta in the Annam area of South-Central Lao PDR. Details ofthis model could not be tested using the present data. The coalescentsimulations were unable to distinguish among more complexpopulation histories (i.e., those involving more than three subpopula-tions or different branch lengths); this maybe due to the use of aparsimony criterion in the reconstruction of gene trees from thesimulated data sets where so many taxa are involved. Parsimony isunlikely to find the correct tree, so with more complex histories the s-values tend to converge on a common value regardless of the true fit ofthe history to the empirical gene tree.

Table 8 shows that major divergence events occurred across therange of N. aperta between 4 and 6.5 Ma. Such divergences maybe aconcerted response to the final Indosinian orogeny around 5 Ma [53].These events increased river gradients, deepened river valleys andeffected some river flow reversals; thereby isolating some taxa anddriving divergence. According to the present results, the radiation ofN. aperta (10–5 Ma) is heterochronous with that of S. mekongi (2.1–1.0 Ma [19]). In contrast, other authors regard the phylogeographies ofthe S. japonicum group taxa to be tightly linked to that of theirintermediate hosts [18,54]. The natural infection rates with S. mekongiin N. aperta effect little selection pressure in that they rarely exceed0.3%. S. japonicum group schistosomes appear to infect at least ninespecies of pomatiopsid snail and transmission occurs in a variety of

etween them, as arrows whose width is proportional to the level of gene-flow (the sumfor each shaded area: CRE, contiguous range expansion; PAN, panmixis; RGE, Restrictednnam region diverges to produce the river ecotype (10–9 Ma); river ecotype enters Laogrowth in the Mekong river, Cambodian N. aperta recolonizes larger rivers of Annambodia and Laos (b1 Ma).

267S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

Table 6Coalescent based estimates for the exponential growth parameters (θe−gt) for the 4- and5-order clades from the NCA)

Clade θ g ln(L) Rank Region(s)

4-1 0.375±0.061 4,442.965±170.490 0.333 1 CLM4-2 0.082±0.010 1,584.185±100.490 0.409 3 CER4-3 0.097±0.009 929.913±50.597 0.967 4 ER/LAR/LMK4-4 0.052±0.011 185.414±53.392 0.003 6 LAR/LMK4-5 0.019±0.004 107.624±60.401 0.019 7 CER/LAR/LMK4-6 0.454±0.063 2,858.983±105.990 0.771 2 LMK4-7 0.024±0.004 46.789±38.495 0.006 8 LAR/LMK/NSP4-8 0.021±0.007 520.048±186.411 0.009 5 NSP4-9 0.007±0.002 75.636±58.333 0.005 9 NSP5-1 0.139±0.015 502.724±46.684 0.304 1 CER/CLM/LAR/LMK5-2 0.122±0.011 501.698±61.234 0.045 2 CER/LAR/LMK5-3 0.046±0.005 55.945±26.660 0.021 3 LAR/LMK/NSP

Maximum likelihood values are given for θ and g±SD.

Table 7Composition of subpopulations in terms of clades and main regions involved

Subpopulation Composition Region(s)

502 bp 4-4, 4-5 Attapeu: (Xe Kong), Laotian Mekong river503 bp 4-6, 4-7, 4-8,

4-9Bolovens: Khong Island area, springs and streams ofNorth-Central Laos

504 bp 4-1, 4-2, 4-3 Kontum: Cambodian rivers (Mekong & eastern)

Table 8Maximum likelihood estimates for dates of key mutations on the gene tree forNeotricula aperta

Phylogenetic event Date Likelihood

Divergence of LAR as a monophyletic clade 10.124±2.960 0.560Divergence of NSP monophyletic clade 9.281±3.011 0.089Divergence of CLM monophyletic clade 6.539±1.623 0.100Divergence of NSP clade into subclades 5.695±2.337 0.005Divergence of Xe Bang Fai river clade from Thai Mekongriver clade or vice versa

5.273±2.282 0.003

Divergence events within LAR clade 5.273±2.667 0.002Divergence of NSP clade into subclades 4.640±1.418 0.722Divergence of NSP clade into subclades 4.008±1.305 0.090Major phylogenetic events within CLM clade 4.008±0.653 0.162

Dates are given in millions of years ±95% confidence interval. See Table 2 for codes.

268 S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

habitats. Consequently, a de-coupling of snail and parasite phyloge-nies is not unexpected.

The predominant direction of historical gene-flow or migrationwas inferred as from the Cambodian Mekong River northwards intothe Xe Kong River valley of central Lao PDR and eastern Cambodia(Fig. 6, tract 1). Some East to West (Sre Pok to Mekong) gene-flowwasalso detected in Cambodia (Fig. 6, tract 2). A further tract of migrationwas from the Cambodian lower Mekong into the Lao Mekong river atKhong Island (tract 3). The NCA, Table 8 (dating) and examination ofintraclade diversities suggest an original population in Central LaoPDR, expanding from the Lao-Thai Mekong River. Consequently, theSouth to North pattern inferred by coalescent methods may representdominant processes occurring within the last 5 Ma. A northwardsmigration tract is against the flow of the modern rivers in the region(except for Fig. 6 tract 2 which could be more recent); however, thePliocene river courses were very different from those of the modernrivers. Marked tectonic upheavals during the Pleistocene effectedmajor course changes, stream captures and flow reversals [55].Consequently, migration rates between Cambodia and Lao PDR haveprobably been low since the early Pleistocene when S. mekongi wasapparently introduced to Cambodia [19]. The NCA shows a biogeo-graphical divide betweenwestern (Lao/Thai) and eastern (eastern Lao/Cambodian) clades; this divide runs along the Bolovens uplift and theuplifted edge of the Khorat Basin (Fig. 5). The NCA can reflect olderpatterns and processes than the coalescent methods; the gene-flowfrom Cambodia to Attapeu (tract 1) is probably a more recentrecolonization of the larger Annam rivers (after the expansion of theriver ecotype in Cambodia b5 Ma) (see Fig. 5 caption for more detail).The lack of more recent gene-flow from Cambodia to Lao PDR couldexplain the absence of S. mekongi from apparently suitable transmis-sion foci (where N. aperta is present at high population densities)across Lao PDR, as a result of history rather than ecology. Thesuggestion is that S. mekongi has not yet had time to migrate into LaoPDR and colonize N. aperta populations beyond Khong Island becausebiogeographical barriers between the two countries have inhibitedand slowed colonization. Such barriers are the Dangrek Escarpment,which runs along the border East of Khong Island, and the MesozoicKontum massive lying between Cambodia, Vietnam and southern LaoPDR. Table 4 shows high levels of gene-flow between clade 4-6 and 4-5, but, as these two clades abut just North of Khong Island, it is likelythat this gene-flow is occurring along the clade borders. The stochasticnature of the coalescent model and the sample size used in thepresent study led to wide confidence intervals for the gene-flowparameters and dates estimated here; however, the general trendswere so marked (e.g. the tracts in Fig. 6 and N2-fold differences ingrowth rates), and/or were supported by the results of more than oneanalysis (using different approaches), that they can be consideredreliable indicators. Interestingly, there is moderate gene-flow between

the Mekong River clades and the entirely spring dwelling clade 4-8(Kong Lor) of northern Lao PDR. Clade 4-8 also exchanges genes/individuals with clade 4-7 at high rates (especially in a southerlydirection, Fig. 6 tract 5); the exchange is probably with clade 4-7populations around the Nam Yom River (note, Fig. 6 shows onlyapproximate clade boundaries and does not show this northwardsextension of clade 4-7 along the Xe Bang-Fai River system).

The largest estimated Ne values were for the lower Mekong Riverclades 4-6 and then 4-1 (these being approximately four times greaterthan the next largest clade, 4-3). The eastern Cambodian riverpopulations and those of the upper Xe Kong had the next highest Ne

estimates, followed by the Xe Bang-Fai river system and the ThaiMekong River. The northernmost clades, namely 4-7, 4-8 and 4-9, hadthe smallest estimated Ne values (around 5% of that for clade 4-6,down to 1.5% for clade 4-9), see Fig. 5. The findings of even thispreliminary study suggest that the coalescent may be used to estimatesnail populations in areas that are inaccessible and difficult to surveydirectly (such as Bolikhamxai Province in Lao PDR). A general guide tothe sizes of snail populations in a river system can be obtained from asingle population sample from that river system.

4.2. Public health implications and summary of the findings

The radiation of S. mekongi appears to be independent of that ofN. aperta because the range of estimated divergence dates for theparasite (i.e., within the last 3 Ma [19]) shows no overlap with that forthe radiation of N. aperta estimated here (13 to 5 Ma for theestablishment of all the major (monophyletic) clades, after includingconfidence intervals). The dating for the radiation of N. aperta agreeswith the post mid-Miocence time-frame suggested by Davis [18]. Thelargest and fastest growing N. aperta populations appear to be in theMekong River. The Lao Mekong River represents the largest followedby the Cambodian Mekong and eastern Cambodian rivers; the fastestgrowing appears to be the Cambodian Mekong, followed by the LaoMekong and the Cambodian eastern rivers. In contrast, the growthrate and estimated Ne for the northern Lao (including spring dwelling)populations and Central Lao populations were much smaller (b10% ofthose for the other group of populations). The only exception to thiswas clade 4-8, a spring dwelling population near to the constructionsite of the Nam Theun II dam project, which showed a high growth

269S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

rate. This observation, together with that of high levels of gene-flow(in a region of otherwise restricted gene-flow) between clade 4-8 andpopulations along the Xe Bang-Fai and Nam Yom rivers (the dischargechannel for the dam), requires further investigation. The possibledetection of unusual population expansion andmigration rates amongN. aperta at the dam site has certain public health implications. Theobservation of a marked expansion in the snail populations at theS. mekongi transmission foci along the Mekong River in Cambodiahighlight the need for snail control and suggest that control ofS. mekongi will require continual efforts in the face of a large andhistorically fast growing, panmictic, snail population.

The findings of a predominant South to North pattern of migration,panmixia in Cambodia, range expansion in the Lao Mekong River, andthe relative lack of gene-flow in Central Lao PDR, suggest that theexpansion of the river ecotype ofN. aperta (which transmits S. mekongiin nature) began in Cambodia and is progressing northwards. Thewidespread occurrence of N. aperta across Lao PDR, as reported here,also suggests that ecological conditions in Lao PDR are suitable forS. mekongi transmission and that the absence of S. mekongi from mostof Lao PDR is due to historical processes. For example, the post-Pliocene rate of exchange of individuals from infected populations ofCambodia has been so low that there has not been sufficient time forS. mekongi to invade Lao PDR; however, migration rates appear to havebeen high at some point in the history of N. aperta, most likely b4 Ma(the time by which most isolating events appear to have occurred, seeTable 8). Further study is required into this matter because it impliesthat the future emergence of S. mekongi across Lao PDR is possible, andthat disease surveillance efforts in Lao PDR need to be enhanced inareas previously assumed to be free from the risk of schistosomiasis. Inaddition, the recognition of a spring-dwelling ecotype of N. aperta inLao PDR implies a new dimension to the S. mekongi controlprogramme. If N. aperta can inhabit small streamlets and pools, thepotential range of S. mekongi is much greater than previously thought.The challenge for disease surveillance is also greater as habitatsmay behidden around villages or forests and the site of transmission wherehuman cases are discovered will be much more difficult to determine.Human exposure patterns will also differ, exposure along the MekongRiver is concentrated among those involved in fishing, laundry etc.;however, transmission via the spring dwelling ecotype would affectthe whole community, with an exposure pattern similar to that ofS. malayensis which is transmitted by snails living in springs andseepages [56,57]. The implications for public health are serious andfurther work is urgently required into the molecular ecology of bothS. mekongi and N. aperta.

Acknowledgement

This work was supported by a Wellcome Trust Project Grant (No.068706) to SWA.

References

[1] Dupont-Vic BE, Soubrane J, Halle B, Richir C. Bilharziose à forme hépato-spléniquerévélée par une grande hématémèse. Bull Mém Soc Méd Hôp Paris 1957;73:933–94.

[2] Harinasuta C, Kruatrachue M. The first recognised endemic area of bilharziasis inThailand. Ann Trop Med Parasitol 1962;56:314–5.

[3] Audebaud G, Tournier-Lasserve C, Brumpt V, Jolly M, Mazaud R, Imbert X, et al.Première cas de bilharziose humaine observé au Cambodge (région de Kracheh).Bull Soc Pathol Exot Filiales 1968;61:778–84.

[4] Sornmani S. Schistosomiasis in Thailand. In: Harinasuta C, editor. Proceedings ofthe fourth Southeast Asian seminar on parasitology, tropical medicine, schistoso-miasis and other snail transmitted helminthiases 22-27 February 1969. Manila:SEAMEO TROPMED; 1969. p. 71–4.

[5] Ohmae H, Sinuon M, Kirinoki M, Matsumoto J, Chigusa Y, Socheat D, et al.Schistosomiasis mekongi: from discovery to control. Parasitol Int 2004;53:135–42.

[6] Sinuon M, Tsuyuoka R, Socheat D, Odermatt P, Ohmae H, Matsuda H, et al. Controlof Schistosoma mekongi in Cambodia: results of eight years of control activities inthe two endemic provinces. Trans R Soc Trop Med Hyg 2007;101:34–9.

[7] Davis GM, Kitikoon V, Temcharoen P. Monograph on “Lithoglyphopsis” aperta, thesnail host of Mekong river schistosomiasis. Malacologia 1976;15:241–87.

[8] Attwood SW, Kitikoon V, Southgate VR. Infectivity of a Cambodian isolate ofSchistosoma mekongi to Neotricula aperta from Northeast Thailand. J Helminthol1997;71:183–7.

[9] Attwood SW. Schistosomiasis in the Mekong region: epidemiology and phylogeo-graphy. Adv Parasitol 2001;50:87–152.

[10] Strandgaard H, Johansen MV, Pholsena K, Teixayavong K, Christensen NO. The pigas a host for Schistosoma mekongi in Laos. J Parasitol 2001;87:708–9.

[11] Matsumoto J, Muth S, Socheat D, Matsuda H. The first reported cases of canineschistosomiasis mekongi in Cambodia. Southeast Asian J Trop Med Public Health2002;33:458–61.

[12] Attwood SW, Upatham ES, Southgate VR. The detection of Schistosoma mekongiinfections in a natural population of Neotricula aperta at Khong Island, Laos, andthe control of Mekong schistosomiasis. J Moll Stud 2001;67:400–5.

[13] Attwood SW, Campbell I, Upatham ES, Rollinson D. Schistosomiasis in the Xe Kongriver of Cambodia: the detection of Schistosoma mekongi in a natural population ofsnails and observations on intermediate host distribution. Ann Trop Med Parasitol2004;98:221–30.

[14] Attwood SW, Upatham ES. A new strain of Neotricula aperta found inKhammouanne Province, central Laos, and its compatibility with Schistosomamekongi. J Molluscan Stud 1999;65:371–4.

[15] Emerson BC, Paradis E, Thébaud C. Revealing the demographic histories of speciesusing DNA sequences. Trends Ecol Evol 2001;16:707–16.

[16] Davis GM. Different modes of evolution and adaptive radiation in the Pomatiopsi-dae (Prosobranchia: Mesogastropoda). Malacologia 1981;21:209–62.

[17] Attwood SW, Ambu S, Meng XH, Upatham ES, Xu FS, Southgate VR. Thephylogenetics of triculine snails (Rissooidea: Pomatiopsidae) from south-eastAsia and southern China: historical biogeography and the transmission of humanschistosomiasis. J Mollusca Stud 2003;69:263–71.

[18] Davis GM. Snail hosts of Asian Schistosoma infecting man: evolution andcoevolution. In: Bruce JI, Sornmani S, Asch HL, Crawford KA, editors. The MekongSchistosome: Malacol Rev; 1980. p. 195–238. Suppl. 2.

[19] Attwood SW, Fatih FA, Upatham ES. A study of DNA-sequence variation amongSchistosoma mekongi (Trematoda: Digenea) populations and related taxa;phylogeography and the current distribution of Asian schistosomiasis. PLoS NeglTrop Dis 2008;2(3):e200, doi:10.1371/journal.pntd.0000200.

[20] Brandt RAM. The non-marine mollusca of Thailand. ArchMollusk 1974;105:1–423.[21] Foster PG. Modelling compositional heterogeneity. Syst Biol 2004;53:485–95.[22] Posada D, Crandall KA. Modeltest: testing the model of DNA substitution.

Bioinformatics 1998;14:817–8.[23] Huelsenbeck JP. Mr. Bayes: Bayesian inference of phylogeny. Department of

Biology. Rochester, NY: University of Rochester; 2000.[24] Excoffier L, Smouse PE, Quattro JM. Analysis of molecular variance inferred from

metric distances among DNA haplotypes: application to human mitochondrialDNA restriction data. Genetics 1992;131:479–91.

[25] Schneider S, Roessli D, Excoffier L. Arlequin, Version 2.0: a software for populationgenetics data analysis. Genetics and Biometry Laboratory. Geneva: University ofGeneva; 2000.

[26] Rogers AR, Harpending HC. Population growth makes waves in the distribution ofpairwise genetic distances. Mol Ecol 1992;9:552–69.

[27] Harpending HC. Signature of ancient population growth in a low-resolutionmitochondrial DNA mismatch distribution. Hum Biol 1994;66:591–600.

[28] Nei M. Evolutionary change of nucleotide sequences. Molecular evolutionarygenetics. New York: Columbia University; 1987. p. 64–110.

[29] Tajima F. Statistical methods for testing the neutral mutation hypothesis by DNApolymorphism. Genetics 1989;123:585–95.

[30] Rozas J, Rozas R. DnaSP version 3: an integrated program for molecular populationgenetics and molecular evolution analysis. Bioinformatics 1999;15:174–5.

[31] Edwards SV, Beerli P. Perspective: gene divergence, population divergence, and thevariance incoalescence time inphylogeographic studies. Evolution 2000;54:1839–54.

[32] Attwood SW, Fatih FA,Mondal MMH, AlimMA, Fadjar S, Rajapakse RP, et al. A DNA-sequence based study of the Schistosoma indicum (Trematoda: Digenea)group; population phylogeny, taxonomy and historical biogeography. Parasitology2007;134:2009–20.

[33] Templeton AR, Crandall KA, Sing CF. A cladistic analysis of phenotypic associationswith haplotypes inferred from restriction endonuclease mapping and DNAsequence data. III. Cladogram estimation. Genetics 1992;132:619–33.

[34] Clement M, Posada D, Crandall KA. TCS: a computer program to estimate genegenealogies. Mol Ecol 2000;9:1657–9.

[35] Swofford DL. PAUP⁎ Phylogenetic Analysis Using Parsimony (and other methods).Sunderland, Massachusetts: Sinauer Associates; 2002.

[36] Posada D, Crandall KA, Templeton AR. Nested clade analysis statistics. Mol EcolNotes 2006;6:590–3.

[37] Templeton AR, Sing CF. A cladistic analysis of phenotypic associations withhaplotypes inferred from restriction endonuclease mapping. IV. Nested analyseswith cladogram uncertainty and recombination. Genetics 1993;134:659–69.

[38] Crandall KA, Templeton AR, Sing CF. Intraspecific phylogenetics: problems andsolutions. In: Scotland RW, Siebert DJ, Williams DM, editors. Models in PhylogenyReconstruction. Oxford: Clarendon Press; 1994. p. 273–97.

[39] Posada D, Crandall KA, Templeton AR. GeoDis: a program for the cladistic nestedanalysis of the geographical distribution of genetic haplotypes. Mol Ecol2001;9:487–8.

[40] Kuhner MK, Yamato J, Felsenstein J. Maximum likelihood estimation of populationgrowth rates based on the coalescent. Genetics 1998;149:429–34.

[41] Beerli P, Felsenstein J. Maximum-likelihood estimation of migration rates andeffective population numbers in two populations using a coalescent approach.Genetics 1999;152:763–73.

270 S.W. Attwood et al. / Parasitology International 57 (2008) 256–270

[42] Knowles LL, MaddisonWP. Statistical phylogeography. Mol Ecol 2002;11:2623–35.[43] Slatkin M, Maddison WP. A cladistic measure of gene flow inferred from the

phylogenies of alleles. Genetics 1989;123:603–13.[44] Maddison WP, Maddison DR. Mesquite: a modular system for evolutionary

analysis; 2007. Version 2.0. http://mesquiteproject.org. Vancouver.[45] Griffiths RC, Tavaré S. Ancestral inference in population genetics. Stat Sci

1994;9:307–19.[46] Attwood SW. A demographic analysis of y-Neotricula aperta (Gastropoda:

Pomatiopsidae) populations in Thailand and southern Laos, in relation to thetransmission of schistosomiasis. J Molluscan Stud 1995;61:29–42.

[47] Xia X, Xie Z, Salemi M, Chen L, Wang Y. An index of substitution saturation and itsapplication. Mol Phylogenet Evol 2003;26:1–7.

[48] Rogers AR. Genetic evidence for a Pleistocene population explosion. Evolution1995;49:608–15.

[49] Attwood SW. The effect of substratum grade on the distribution of the freshwatersnail y-Neotricula aperta (Temcharoen), with notes on the sizes of particlesingested. J Molluscan Stud 1995;61:133–8.

[50] Davis GM. The origin and evolution of the gastropod family Pomatiopsidae, withemphasison theMekongriverTriculinae.AcadNat SciPhilad,Monogr1979;20:1–120.

[51] Attwood SW, Upatham ES, Zhang YP, Yang ZQ, Southgate VR. A DNA-sequencebased phylogeny for triculine snails (Gastropoda: Pomatiopsidae: Triculinae),intermediate hosts for Schistosoma (Trematoda: Digenea): phylogeography andthe origin of Neotricula. J Zool 2004;262:47–56.

[52] Carstens BC, Degenhardt JD, Stevenson AL, Sullivan J. Accounting for coalescentstochasticity in testing phylogeographical hypotheses: modelling Pleistocenepopulation structure in the Idaho giant salamander Dicamptodon aterrimus. MolEcol 2005;14:255–65.

[53] Rost KT. Pleistocene paleoenvironmental changes in the high mountain ranges ofcentral China and adjacent regions. Quat Int 2000;65/66:147–60.

[54] Davis GM, Wilke T, Zhang Y, Xu XJ, Qiu CP, Spolsky C, et al. Snail-Schistosoma,Paragonimus interactions in China: population ecology, genetic diversity,coevolution and emerging diseases. Malacologia 1999;41:355–77.

[55] Hutchinson CS. In: Charnock H, Conway Morris S, Dewey JF, Navrotsky A, OxburghER, et al, editors. Geological evolution of South-east Asia. Oxford: Clarendon Press;1989.

[56] Greer GJ, Ambu S, Davis GM. Studies on the habitat, distribution and schistosomeinfection of Robertsiella spp., snail hosts for a Schistosoma japonicum-likeschistosome in Peninsular Malaysia. Trop Biomed 1984;1:85–93.

[57] Attwood SW, Lokman HS, Ong KY. Robertsiella silvicola, a new species of triculinesnail (Caenogastropoda: Pomatiopsidae) from peninsular Malaysia, intermediatehost of Schistosoma malayensis (Trematoda: Digenea). J Molluscan Stud2005;71:379–91.

[58] Templeton AR. Nested clade analysis of phylogeographic data: testing hypothesesabout gene flow and population history. Mol Ecol 1998;7:381–97.