THE PHYLOGENETICS OF TRICULINE SNAILS
(RISSOOIDEA:POMATIOPSIDAE) FROM SOUTH-EAST ASIA
AND SOUTHERN CHINA: HISTORICAL BIOGEOGRAPHY AND
THE TRANSMISSION OF HUMAN SCHISTOSOMIASIS
S. W. ATTWOOD 1 , S. AMBU 2 , X.-H. MENG 3 , E. S. UPATHAM 4 , F.-S. XU 3 AND
V. R. SOUTHGATE 1
1
Wolfson Wellcome Biomedical Laboratories, Department of Zoology, The Natural History Museum, London SW7 5BD, UK; 2Institute for Medical Research,
Jalan Pahang, Kuala Lumpur, Malaysia; 3Sichuan Institute of Parasitic Diseases, Chengdu 610041, PR China; and 4Department of Biology, Faculty of Science,
Mahidol University, Bangkok 10400, Thailand
(Received 13 August 2002; accepted 19 February 2003)
ABSTRACT
Partial DNA sequences were examined for one nuclear (18S rRNA) and two mitochondrial (16S rRNA
and CO1) loci for six species of pomatiopsid snail (Gastropoda:Rissooidea:Pomatiopsidae) from southeast Asia and south-west China. Fresh field samples were collected for the following taxa: Neotricula aperta
(Triculinae:Pachydrobiini) from southern Laos; Neotricula burchi from northern Thailand; Oncomelania
hupensis robertsoni (Pomatiopsinae:Pomatiopsini) from south-west China; Robertsiella sp. (Pachydrobiini)
from West Malaysia; Tricula bollingi (Triculinae:Triculini) from northern Thailand; and Tricula hortensis
from south-west China. Sequences taken from GenBank for Gammatricula fujianensis (Pachydrobiini)
were also used. This represents the first published DNA sequence data for N. burchi and Robertsiella. With
the exception of N. burchi, all of these taxa transmit Schistosoma in nature; N. aperta, O. h. robertsoni
and Robertsiella transmit Schistosoma to humans. All of the above taxa were found to be homogenetic
(i.e. showed no sequence variation) at the 18S locus. Phylogenies were estimated using a fully optimized
model of nucleotide substitution and either a maximum likelihood or Bayesian method. Good congruence was observed between the phylogenies resulting from the two different methods. The 16S and CO1
trees showed the same topology except for the relationships between G. fujianensis, T. hortensis and the
other taxa. The data confirmed the congeneric status of N. aperta and N. burchi; the implications of this for
the choice of historical biogeographical model for the Pachydrobiini are discussed.
INTRODUCTION
The Pomatiopsidae Stimpson, 1865 are conservative rissooidean
snails, eight of which are known to act as intermediate host for
species of Schistosoma Weinland, 1858 (Trematoda:Digenea; see
Davis, 1992). The Pomatiopsidae appear to have arisen on the
Indian Plate and colonized south-east Asia, from northeast
India, during the mid-Miocene (Davis, 1979). Today the more
generalized Pomatiopsidae inhabit streams and minor rivers,
draining hillsides and other highland areas. This ecological
habit may reflect the ancestral habitat during the early radiation
of this clade in the highlands of northern India and Burma. The
habitat requirements of these taxa, which appear to preserve
past biogeographical patterns, have led to their use in historical
biogeographical studies, not only of Rissooidea, but also of
Schistosoma (see Davis, 1979, 1992; Attwood, 2001; Attwood &
Johnston, 2001). In spite of the importance of this group, taxonomic questions remain at all systematic levels, and the purpose
of the present study was to address certain problems at the genus
and subfamily levels.
The Pomatiopsidae comprise two subfamilies, the Pomatiopsinae Stimpson, 1865 and the Triculinae Annandale, 1924. The
Pomatiopsinae include Oncomelania hupensis Gredler, 1881, the
intermediate host of Schistosoma japonicum Katsurada, 1904 a
parasite of humans in central and southern China (and other
areas, see Rollinson & Southgate, 1987). The Pomatiopsinae
have a Gondwanan distribution. The extant Triculinae are found
along a tract running from northern India into southern China
and south-east Asia. The Triculinae comprise three tribes (see
Correspondence: S. W. Attwood; e-mail: [email protected]
J. Moll. Stud. (2003) 69: 263–271
Davis, Chen, Wu, Kuang, Xing, Li, Liu & Yan, 1992); these are the
Jullieniini, Pachydrobiini and Triculini. The Triculinae include
Neotricula aperta (Temcharoen, 1971) (Pachydrobiini) the intermediate host of Schistosoma mekongi Voge, Buckner & Bruce, 1978,
a parasite of humans along the Mekong River of Laos and
Cambodia (see Attwood, 2001). In addition to N. aperta, several
other Triculinae are known to act as intermediate host for
Schistosoma species Robertsiella kaporensis Davis & Greer, 1980
(Pachydrobiini) transmits S. malayensis Greer, Ow-Yang & Yong,
1988 in peninsular Malaysia; this is a parasite of rodents but also
infects the aboriginal peoples of the region. Tricula bollingi Davis,
1968 (Triculini) transmits S. ovuncatum Attwood, 2002 (and possibly also a sympatric strain of S. sinensium) to rodents in northern
Thailand (Attwood, Panasoponkul, Upatham, Meng & Southgate,
2002a). A Tricula Benson, 1843 species from Sichuan, China
(studied here) transmits another rodent Schistosoma, S. sinensium
Bao, 1958, on the Yangtze River plain (Bao, 1958; Attwood et al.,
2002a).
The small size of triculine snails (often <2 mm in length) leads
to a lack of clearly variable morphological characters and to technical difficulties in the laboratory. In many cases, type material,
which for some taxa dates back to the nineteenth century, is
either inadequate (for example, only shells) or inaccessible. In
addition, triculine taxa are often distinguished on the basis of a
relatively small number of morphological characters; these problems, together with a high prevalence of convergent evolution
and a relatively high degree of intraspecific variation among
anatomical characters, call for corroboration of taxonomic
hypotheses for the Triculinae using data other than anatomical
differences. The present work attempts to do this using DNA
© The Malacological Society of London 2003
S. W. ATTWOOD ET AL.
N. burchi as a geographical isolate associated with the Ping River
of northern Thailand is more difficult to explain (Attwood, 2001)
and, in historical biogeographical terms, it would be simpler to
make a case for Tricula burchi than N. burchi. Neotricula, as defined
by Davis, Subba Rao & Hoagland (1986), differs consistently from
Tricula in only four main character states, one of which can occur
to some degree in Tricula and two of which cannot be objectively
scored (for example, the narrowing of the bursal duct in
Neotricula). In effect, the only reliable character distinguishing
the two genera is the state of the spermathecal duct in the female;
in Tricula it enters the pericardium, whereas in Neotricula it does
not. Furthermore, T. bollingi and N. burchi are found in near sympatry in Chiang-Mai Province, northern Thailand (Fig. 1). N.
burchi has not been found outside the Pliocene Ping River
drainage, and no other Tricula species is found in Thailand or
northern Laos. In view of the fact that the case for N. burchi is relevant to the choice of historical biogeographical model for the
south-east Asian Triculinae, and that a particular character state
of the spermathecal duct could evolve independently in different
lineages, it was decided to include N. aperta, N. burchi and T. bollingi
in this study. Robertsiella was included for similar reasons;
this taxon is only found in peninsular Malaysia (Fig. 1) and yet
morphological studies place its closest relatives in Hunan
Province, China, over 5000 km away (Davis et al., 1992). The
species referred to below as Robertsiella sp. was originally collected
by Greer, Ambu & Davis (1984), from Baling, peninsular Malaysia,
and was found to shed cercariae of S. malayensis. However, these
authors noted morphological differences between the Baling
taxon and the other two species of Robertsiella (R. gismani and
R. kaporensis). Yong et al. (1985) reported large genetic distances
(based on allozyme variation) between the Baling taxon and the
sequence data, as such data are still greatly limited for this group.
The choice of taxa for study was designed to permit the assessment of relationships that were particularly relevant to historical
biogeographical modelling for the Triculinae.
The first studies of genetic variation among the Triculinae
were based on allozyme differences (Yong, Ooi, Greer, Lai &
Ow-Yang, 1985; Staub, Woodruff, Upatham & Viyanant, 1990;
Davis, Chen, Xeng, Yu & Li, 1994). These biochemical studies
were soon followed by studies of DNA sequence variation among
the Pomatiopsidae, which were at first restricted to the O. hupensis
complex and involved the mitochondrial (mt) cytochrome-c
oxidase subunit 1 (CO1) and cytochrome-b genes (Hope &
McManus, 1994; Spolsky, Davis & Yi, 1996; Rosenberg, Tillier,
Tillier, Kuncio, Hanlon, Masselot & Williams, 1997). Partial
sequences are now available for the mt16S, CO1 and nuclear 18S
rRNA genes of Oncomelania hupensis robertsoni Bartsch, 1946 and
Erhaia jianouensis (Liu & Zhang, 1979), both Pomatiopsinae, and
for the Triculinae Lacunopsis sp. Deshayes, 1876, Tricula sp. and
Gammatricula spp. (Davis & Liu, 1990) (see Davis, Wilke, Spolsky,
Zhang, Xia & Rosenberg, 1998; Wilke, Davis, Gong & Liu, 2000;
Wilke, Davis, Falniowski, Giusti, Bodon & Szarowska, 2001). The
only data for south-east Asian Triculinae were provided by
Attwood & Johnston (2001) who reported partial CO1 sequences
for N. aperta of Cambodia, Laos and Thailand and for T. bollingi of
northern Thailand.
Study of the Triculinae reveals taxonomic and biogeographical
problems at the generic level. The presence of N. aperta in the
Mekong River of Laos and Cambodia was explained as a result of
dispersal from southern China, where most of the major radiations (i.e. those with over 10 endemic species) of the Triculinae
are now found (Davis et al., 1992). However, the presence of
Figure 1. The major rivers draining the eastern margins of the Tibetan Plateau, and the collecting sites for the present study. The symbol * marks the position
of the lake at Dali. 1, Tricula hortensis, Han-Wang, Sichuan, PR China; 2, Oncomelania hupensis, Mianzhu, Sichuan; 3, Tricula bollingi and Neotricula burchi, Ping
River valley of Chiang-Mai Province, northern Thailand; 4, Neotricula aperta, Khong Island, Mekong River, southern Laos; 5, Robertsiella sp., Ketil River system,
Baling, Kedah State, peninsular Malaysia. Scale approximate.
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an examination of congruence between phylogenies based on
different loci and using different phylogenetic methods.
The CO1 locus was chosen because it was expected to show
sufficient variation, even between congeneric or sister taxa. Attwood & Johnston (2001) reported haplotype diversities as high as
0.029 within N. aperta for the same CO1 locus. The mt16S gene
was chosen because it has previously been used successfully to
assess family level relationships among gastropods (e.g. Remigio
& Blair, 1997) and among Pomatiopsidae (Wilke et al., 2000). The
mtDNA loci, with their maternal pattern of inheritance, and
therefore smaller effective population size, may be expected to
evolve more rapidly, and be better suited to reconstructing more
recent biogeographical events and divergences. In view of this the
nuclear 18S rRNA gene was also included in order to resolve any
deeper phylogenetic divergences. Several pomatiopsid taxa are
known to be homogenetic at certain regions of the 18S gene
(Wilke et al., 2001). However, Stothard, Brémond, Andriamaro,
Loxton, Sellin, Sellin & Rollinson (2000) reported considerable
intraspecific variation at the variable domains (V1 and V2) of the
18S rRNA gene for Lymnaea natalensis Krauss, 1848 (Gastropoda:
Pulmonata); the region examined here corresponds to positions
91–385 of their sequence (AF192273 in GenBank).
Total genomic DNA was used as a template for PCR amplification on a Progene thermal cycler (MWG), using standard
PCR conditions as described in Clackson, Güssow & Jones (1991).
Unincorporated primers and nucleotides were removed from
PCR products using the QIAQuick PCR purification kit (QIAGEN). Sequences were determined directly from the PCR
templates by thermal cycle sequencing using Big Dye fluorescent
dye terminators and an ABI 377 automated sequencer (PerkinElmer), using procedures recommended by the manufacturers.
DNA extracts were not pooled and one DNA sequence represented one snail. Sequences were assembled and aligned using
Chromas (McCarthy, 1996) and ClustalX (Thompson, Higgins
& Gibson, 1994). DNA sequences for both the forward and
reverse strands were aligned and compared to confirm accuracy.
Negative controls were run alongside all PCRs, and results from
different DNA extractions, PCRs and sequencing reactions (performed at least 1 week apart) for the same OTUs (operational
taxonomic units) were checked for agreement.
other two species, when compared with the distance between the
two named species. The morphology of this taxon has yet to be
examined fully. Tricula hortensis Attwood & Brown, 2003 is a new
species recently described in Attwood, Brown, Meng & Southgate
(2003). The pomatiopsine Oncomelania hupensis robertsoni was
sampled to allow comparisons with the triculine taxa, and Gammatricula fujianensis (Liu, Zhang & Wang, 1983) (Pachydrobiini)
sequences were taken from GenBank to afford better comparisons between the Pachydrobiini and Triculini.
MATERIAL AND METHODS
Sampling
The sampling area encompassed four countries and six different
collecting sites (Fig. 1). Table 1 gives details of sampling sites
and dates of collection. The snails were identified on the basis of
general shell form, radular characters, ecological habit, and gross
dissection of pallial and reproductive structures. All snails, with
the exception of the Robertsiella sp., were collected by the first
author and were readily identified. The Robertsiella sample was
collected by a team from the Institute of Medical Research in
Kuala Lumpur from the locality at which Greer et al. (1984) collected a new species of Robertsiella that has not yet been named.
The sample in the present study appears to be from the same
population as sampled by Greer et al. (1984); this snail was
believed to transmit S. malayensis to rodents in the area (Ambu,
Greer & Shekhar, 1984). Fresh samples were fixed in 100%
ethanol directly in the field. Samples for N. aperta, N. burchi and
T. bollingi were also taken in 10% neutral formalin to aid identification.
DNA amplification and sequencing
The snails were gently crushed and the body separated from
the shell. The gut and digestive gland were removed and DNA
extracted from the remainder by standard methods (Winnepenninck, Backeljau & DeWachter, 1993). Selected DNA sequences
were amplified by polymerase chain reaction (PCR). Amplification of a section of the coding region of the mt cytochrome-c
oxidase subunit 1 (CO1) was achieved with the HCO-2198
and LCO-1490 primer pair developed by Folmer, Black, Hoeh,
Lutz & Vrijenhoek (1994), following the recommended cycling
conditions. Part of the mt 16S rRNA gene was amplified using
the primers of Palumbi, Martin, Romano, McMillan, Stice & Grabowski (1991). For the 18S locus, primers SWAM18SF1 (5-gaatggctcattaaatcagtcgaggttccttagatgatccaaatc-3) and SWAM18SR1
(5-atcctcgttaaagggtttaaagtgtactcattccaattacggagc-3) were designed,
using an alignment of GenBank sequences AF21908 (Tricula) and
AF212906 (O. h. robertsoni; see Wilke et al., 2000). No published
data were available for two of the taxa studied and only CO1 data
were available for a further two. Consequently, the aim was to
amplify shorter sequences at a variety of loci; the data would then
be more useful in determining the most phylogenetically informative loci for these taxa. The current approach also allows
Phylogeny reconstruction
Data (as consensus sequences) for the OTUs sampled were
grouped together into sets of aligned sequences of equal length,
so that all taxa were represented in each set. Sample sizes, upon
which the consensus sequences for each OTU were based, are
given in Table 2. No intrataxon variation was found in any OTU
sample. The 18S sequences were found to be the same for all the
taxa studied and further analyses were not performed on these
data. An outgroup sequence, taken from GenBank (AF212898,
Wilke et al., 2001), for Hydrobia acuta (Draparnaud, 1805) (Rissooidea:Hydrobiidae) collected in France, was added to the CO1
data. A similar out-group was added to the 16S data set; this was
GenBank sequence AY010324 (Wade, Mordan & Clarke, 2001)
for a Hydrobia sp. Hartmann, 1821. The CO1 sequence AF213342,
Table 1. Snail collecting sites, dates and species for taxa sampled during the present study.
Taxon
Collecting site
Date
Co-ordinates
Neotricula aperta
Ban Hat Xai Khoun, Mekong River, Laos
28/04/01
14°630 N; 105°5145 E
Neotricula burchi
Chiang-Dao Cave, Chiang-Mai, Thailand
23/05/01
19°2330 N; 98°560 E
Oncomelania hupensis robertsoni
Mianzhu, Sichuan, PR China
14/04/00
30°40 N; 104°830 E
Robertsiella sp.
Baling, Kedah, Malaysia
20/03/01
5°4230 N; 100°5815 E
Tricula bollingi
Fang, Chiang-Mai, N Thailand
22/03/00
19°3830 N; 99°520 E
Tricula hortensis
Han-Wang, Sichuan, China
12/04/00
30°415 N; 104°815 E
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S. W. ATTWOOD ET AL.
was assessed by bootstrap resampling (5000 replicates). For each
data set a 2-test for intertaxon variation in nucleotide frequency
was performed using PAUP*. The data were tested for (substitution) saturation using plots of the numbers of transitions and
transversions against the ML genetic distance (following DeSalle,
Freedman, Prager & Wilson, 1987). The indications of these plots
were further evaluated using the entropy- based test of Xia, Xie,
Salemi, Chen & Wang (2002) as found in the DAMBE software
package of Xia (1999), which provides a statistical test for saturation. The test was chosen because it was thought more likely to
detect saturation in the present data, where several closely related
species are compared, than randomization or permutation tests,
or the test of Lyons-Weiler, Hoelzer & Tausch (1996). LRTs
were performed in order to compare the maximum likelihoods
obtained under the ML model with and without a molecular
clock enforced; the resulting probability is the probability that
one would be incorrect in rejecting the null hypothesis that there
is no difference in evolutionary rate among taxa in the data set
(Felsenstein, 1988). A Shimodaira–Hasegawa test Shimodaira &
Hasegawa, 1999; see also Goldman, Anderson & Rodrigo, 2000)
was used to compare the log-likelihoods of trees of differing
topologies constructed using the different data sets. The test
was performed using PAUP*, with 1,000 bootstrap replicates.
Statistics relating to polymorphism (see Table 3) were computed
using the DNAsp program of Rozas & Rozas (1999).
for G. fujianensis, reported by Wilke et al. (2000) to GenBank, was
added to the data; this taxon was reportedly collected in Fujian
Province, China. A GenBank sequence G. fujianensis was also
added to the 16S data set (AF212896; Wilke et al., 2000), but this
sequence was 21 bp shorter than the 16S sequences obtained
during the present study. Consequently, it was necessary to trim
the 16S sequences for the other seven taxa (removing three polymorphic sites in the process) in order to achieve complete alignment; the 16S data were analysed both with and without the
G. fujianensis sequence so that the effect (if any) of removal of
some data on the phylogenetic reconstruction could be assessed.
Phylogenetic analysis was undertaken using both a maximum
likelihood (ML) and a Bayesian method. The present data
showed significant variation in the rate of substitution among
sites, together with considerable bias among the six different
types of nucleotide substitution. In such cases, the ML method is
considered more robust than most other commonly used phylogenetic methods, as it makes possible a fully optimized model of
substitution (Nei, 1991). A Bayesian method was used, in addition to a standard ML analysis, as it also allowed the specification
of a full range of substitution model parameters, and the ready
assessment of the credibility or nodal support of each clade in the
overall tree. The Bayesian method was implemented using the
MrBayes version 2.01 program of Huelsenbeck & Ronquist
(2000). The Bayesian approach used Metropolis-coupled MCMC
(see Huelsenbeck, Ronquist & Hall, 2000) and four Markovchains were run simultaneously.
A suitable substitution model was selected using hierarchical
testing of alternative models of nucleotide substitution by mixed
2-test as implemented in Modeltest v. 3.06 (Posada & Crandall,
1998). A general time-reversible ML model was selected for both
the 16S and CO1 data sets; the model accommodated among-site
rate heterogeneity with (the shape of the gamma distribution)
equal to 0.201 for the 16S data (0.208 with G. fujianensis included)
and 0.213 for the CO1 data. In both cases a full matrix of rates for
the six different classes of nucleotide substitution was estimated
under the appropriate model, as were the frequencies of the four
different nucleotides. The model used was a GTR + type model
for the 16S data. However, a GTR + SS model was also tested for
the CO1 data in order to allow for different rates between codon
positions (as a majority of the polymorphic sites observed were
at third codon positions—see Results); this model was found
to be preferable to the GTR + for the CO1 data (–lnL = 2179.06
for GTR + SS and 2339.50 for GTR + ; likelihood ratio test,
LRT, P < 0.0001). Consequently, a GTR + SS model was used for
the CO1 data. Genetic distances quoted are those calculated
under the appropriate ML model using PAUP* version 4.0b10
(Swofford, 2002). Heuristic searches were performed (under
the respective ML model) using PAUP* with random addition
sequence (10 replicates) and tree-bisection-reconnection branch
swapping options in effect. Similarly, for the Bayesian analyses,
separate partitions were defined for each of the three-codon positions in the CO1 data, with partition-specific rates estimated by
MrBayes and other general parameters set as in the ML analysis
(except that in MrBayes the command ‘revmat’ was set to estimate
the parameters of the substitution matrix). Model parameters for
the 16S data were also set as for the ML studies. Log-likelihood
scores for the trees generated using MrBayes were plotted against
generation number (over 20,000 generations) and the generation number where the log-likelihoods first reached a plateau was
noted. Only trees saved after this generation number (i.e. after
the burn-in) were used to produce the consensus tree. Posterior
probabilities were then estimated over 500,000 generations
beyond the assumed point of stationarity. The MrBayes program
was run three times on each data set to reduce the chance that the
search algorithm had been attracted to a suboptimal tree. The
clade probability values generated by MrBayes were used as an
indication of nodal support. In the ML analyses nodal support
RESULTS
Sequence analysis
The data were submitted to GenBank under accession numbers
AF531539–AF531556. Table 3 provides basic statistics for the
mitochondrial loci examined. The taxa were homogenetic at the
18S locus and these data are not examined further here. The
CO1 data appeared most informative; 33% of the sites were polymorphic and, of these, 54% were parsimony informative (i.e.
Table 2. Sample sizes, i.e. the number of snails upon which
each consensus sequence was based.
Locus
___________________
Taxon
CO1
16S
Neotricula aperta
Neotricula burchi
O. h. robertsoni
Robertsiella sp.
Tricula bollingi
Tricula hortensis
9
4
4
6
4
4
10
4
4
5
4
4
18S
9
2
2
4
5
5
Table 3. Statistics relating to each locus used in the phylogenetic analyses:
length of amplicon, discounting primer sequences (L); number of OTUs
(N); number of haplotypes found (Hap); total number of sites excluding
those with alignment gaps (T); number of polymorphic sites, with parsimony
informative sites in parentheses (PS); significance of the 2-test for intertaxon heterogeneity in nucleotide frequency (PN); significance of the likelihood-ratio test for a molecular clock (LRT).
16S with Gammatricula
L (bps)
N
Hap
T
PS
PN
LRT
266
CO1
16S original data
fujianensis
598
8
8
598
198 (106)
0.825
0.101
510
7
7
497
123 (38)
0.999
0.031
490
8
8
475
120 (43)
0.999
0.158
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showed a minimum of two different characters with each character present in more than one taxon) and the remaining 46% were
singletons. At the 16S locus 24% of sites were polymorphic and
31% of these were parsimony informative (24 and 36%, respectively, for the expanded data set, i.e. with G. fujianensis included).
In-group genetic distances across the CO1 data set ranged
from 0.1497 (T. bollingi to T. hortensis) to 0.3848 (O. h. robertsoni to
N. burchi). In-group distances across the 16S data ranged from
0.0484 (N. aperta to N. burchi) to 0.1567 (O. h. robertsoni to
Robertsiella). A LRT for a molecular clock (see Table 3) for the 16S
data (without G. fujianensis) failed to support the hypothesis
that the different lineages had been evolving at the same rate.
Although the results of similar LRTs for the CO1 and expanded
16S data sets did not suggest rejection of the molecular clock
hypothesis, the associated confidence levels were not so high as to
justify application of a clock. Plots of the numbers of transitions
and transversions against genetic distance revealed slight saturation at higher distance levels in the CO1 and 16S (original and
expanded) data sets. However, the test of Xia et al. (2002) suggested that the levels of saturation observed were not likely to affect
the analyses (Iss < Iss.c, P = 0.000 for all three data sets; note, a
statistically insignificant difference here would imply a poor
phylogenetic signal). Examination of polymorphism in the CO1
data (using DNAsp) revealed that 17% of the total number of
polymorphic sites corresponded to first codon positions, 1% to
second positions and 82% to third codon positions. Of the estimated total of 229 mutations, 209 were silent, but 20 led to amino
acid replacements.
Phylogeny reconstruction
The topology of the optimal tree for the CO1 data was the same
irrespective of the phylogenetic method used (ML or Bayesian).
However, the Bayesian method tree showed higher levels of
support for all nodes; the ML tree is not shown here, but Figure
2A shows the Bayesian tree for the CO1 data. The topologies for
the ML and Bayesian trees resulting from the analysis of the
expanded 16S data set were also the same and, again, nodal
support was higher for the Bayesian tree. Similarly, the trees for
the original and expanded data sets showed the same topology
(allowing for the absence of one branch on the former), but
overall levels of nodal support were higher on the tree for the
expanded data set. The Bayesian tree for the original 16S data set
also showed higher levels of overall nodal support. Consequently,
Figure 2. Phylograms resulting from a Bayesian method. A. The tree with the maximum posterior probability for the analysis of the mitochondrial CO1 gene
sequences (outgroup taxon Hydrobia acuta). B. The tree with the maximum posterior probability for the mitochondrial 16S rRNA gene DNA sequences
(outgroup taxon Hydrobia sp.). The numbers assigned to each node represent the posterior probability that the clade to the right is correct. Four simultaneous
Markov Chains were run for 500,000 generations past the apparent point of stationarity.
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Introduction, together with the implications for historical biogeographical reconstructions, called for additional data to confirm the congeneric status of N. aperta and N. burchi. The present
data have provided support for the congeneric status of these
taxa and this necessitates a reconsideration of the historical biogeography of the Triculinae.
Davis (1979) suggested that the ancestors of the Pomatiopsidae arose in Gondwana and were rafted to mainland Laurasia
on the Indian Plate. Pomatiopsid snails are thought to have
colonized southern China and south-east Asia after the collision
of the Indian Plate, during the resulting progressive uplift of
the Tibetan Plateau; the hypothesized route of colonization
was from northeast India and into Tibet and southern China
via the northwest-Burma–Brahmaputra–Upper Irrawaddy corridor which opened around 18 Ma (million years ago; Fig. 1). The
elevation of the Tibetan Plateau (38 Ma) led to the creation of
the Salween, Mekong and Yangtze Rivers. In Yunnan (southern
China) the rivers run close together from their origins in Tibet
(Hall, 1998, 2002). The ancient lakes of Burma and Yunnan,
which lie between the rivers (for example, Dali lake, Fig. 1) are
remnants of bridges between the rivers and contain triculine taxa
as isolated, endemic, populations (Davis, Kuo, Hoagland, Chen,
Yang & Chen, 1983). All three tribes of the Triculinae are represented in each of the three rivers and this led Davis (1979) to cite
northern Yunnan, near the border of the Tibetan Plateau, as the
origin of the clade. The current biogeographical deployment of
N. aperta and N. burchi is, at first sight and for the following reasons, more consistent with the latter being a species of Tricula.
Tricula bollingi is found in northwest Thailand, near Chiang-Mai
(Fig. 1), less than 300 km from the border with Yunnan, and with
the two regions interconnected by the Mekong and Ping River
drainages. It is most likely that proto-T. bollingi evolved from taxa
of the Yunnan lakes region, becoming isolated in the extended
Mekong-Ping River around 1.5 Ma (see Hutchinson, 1989); this
extended river ran from northern Yunnan, through Laos and
into Thailand in the region of Chiang-Mai. Neotricula burchi is
also found near Chiang-Mai and one might expect that Neotricula
was derived from the T. bollingi radiation into Thailand, with
N. aperta evolving from N. burchi-like taxa entering the Mekong
River in northeastern Thailand. In support of this, Brandt (1974)
reported finding N. burchi in the Loei River drainage, which lies
along the northern border of the northeastern provinces of
Thailand. Under this model, N. aperta would have diverged from
the T. bollingi–N. burchi lineage and been isolated in the Mekong
River as this river cut eastwards and southwards into central and
southern Laos and Cambodia (where N. aperta is found today);
this scenario is depicted in Figure 3A. However, neither of the
trees presented here shows the close relationship between
N. burchi and T. bollingi that would be expected if a Mekong–Pingassociated phylogeography for N. aperta were correct.
The question of the phylogeography of N. aperta is an important one because it also relates to the divergence of Schistosoma
mekongi from proto-S. japonicum (the latter being found in southern China) and its current distribution, which is in Cambodia and
southern Laos (Attwood, Upatham, Meng, Qiu & Southgate,
2002b). One solution to this problem may be found in the
‘Red River hypothesis’ of Attwood (2001), which explains how
Neotricula could have crossed the mountain barrier from Hunan,
China (not Yunnan), and into Vietnam and Laos during the late
Pliocene. The theme of this model is that the Red River (which is
the only river to cut through the mountains separating China
from south-east Asia today) would have provided a dispersal corridor from Hunan to Vietnam because this river and the Yangtze
River once flowed along a common course. Proto-N. aperta
may then have arrived in northern Laos directly from Hunan
and colonized the Mekong River. Neotricula burchi would then be
derived from this lineage, evolving from (originally Mekong
River) taxa isolated in the extended Loei–Mekong branch of
the Bayesian tree for the expanded data set is used to represent
the 16S phylogeny here (Fig. 2B).
The clade [Robertsiella (N. aperta, N. burchi)] is found in both
the CO1 and 16S trees (Fig. 2), with levels of nodal support over
90% on the 16S tree and 84–95% on the CO1 tree. The 16S tree
shows G. fujianensis (Pachydrobiini) as basal to the pachydrobiine
clade described above (nodal support 90%). In contrast, the CO1
tree does not support the monophyly of the Pachydrobiini, as
G. fujianensis is shown at the base of the lineage leading to the two
Tricula spp. The two trees also differ in the position of T. hortensis;
the CO1 tree shows this taxon as part of a Gammatricula–Tricula
clade, whilst the 16S tree shows it as lying outside the entire pomatiopsid clade. As the levels of support for the T. hortensis lineage
are low on both trees, the position of this taxon is regarded as
unresolved by these data. Neither the CO1 or 16S tree provides
evidence to question the monophyly of the Triculinae, in that
both trees show O. h. robertsoni (Pomatiopsinae) as basal to or outside the lineage bearing the triculine taxa (except for T. hortensis
on the 16S tree). Using the Shimodaira–Hasegawa-test with the
CO1 data, the 16S tree was not significantly worse than the CO1
tree (P = 0.14). However, using the 16S data, the CO1 tree was
significantly worse than the 16S tree (P = 0.04).
DISCUSSION
Phylogenetics
Irrespective of the locus chosen, the congeneric status of
N. aperta and N. burchi was supported by the present data; this is in
agreement with the taxonomy of Davis et al. (1986). Similarly the
data did not reveal a compelling need to question the monophyly
of the Pomatiopsidae and Triculinae. Although the CO1 tree
did show G. fujianensis as associated with the Tricula clade, the 16S
tree (the statistically more favoured tree) shows this taxon as basal
to the pachydrobiine clade (Fig. 2). The CO1 data are thus at odds
with morphological studies in which Gammatricula is described as
a derived, rather than conserved, Pachydrobiini (see Davis et al.,
1992). The CO1 tree alone might be justification for a reconsideration of the polarity of morphological character change in
the Pachydrobiini. However, the ambiguity arising when the 16S
tree is also considered suggests that any re-evaluation be deferred
until called for by additional data (morphological or molecular).
The position of T. hortensis was not well resolved by the present
data. Blast searches, using the consensus sequences obtained for
T. hortensis in this study, revealed a 99% identity with a partial
CO1 sequence for an unnamed Tricula sp. collected by Davis
et al. (1998). It is likely that the sample taken by Davis et al. (1998)
and T. hortensis here represent the same taxon, particularly as
both were collected from near Chengdu, Sichuan, China. Davis
et al. (1998) also reported difficulty in placing this taxon within
the Pomatiopsidae using CO1 DNA sequence data. Attwood et al.
(2003) described T. hortensis as a conserved triculine snail in that
the female reproductive anatomy possessed features common to
both Triculinae and Pomatiopsinae. The evolutionary position
of this taxon at the base of the triculine clade could explain the
difficulty in resolving its position within the Pomatiopsidae. The
hypothesized rapid and recent radiation of the Triculinae in
south-east Asia and China, which according to Attwood et al.
(2002a, 2003) was a Plio-Pleistocene event, suggests that a fully
resolved phylogeny for these taxa may not be possible even with
mitochondrial loci as used here.
Historical biogeographical implications
Davis et al. (1986) created the new genus Neotricula and transferred
Tricula aperta and Tricula burchi to this genus, while T. bollingi
remained in situ. The limited number of diagnostic characters
available and the lack of useful type material, as mentioned in the
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Figure 3. Pliocene river courses in northern Thailand, Laos and Vietnam, and two possible scenarios for the colonization of south-east Asia by Neotricula.
A. Colonization from northern Burma, via the extended Salween–Ping and Mekong–Loei–Passac Rivers and into southern Laos. B. Colonization from southern China (Hunan) and northern Vietnam (via the Pliocene Red River system), with subsequent dispersal eastwards to the Mekong–Ping extended drainage.
These rivers are assumed to have flowed westwards prior to the tilting of the Khorat Plateau, which reversed their flow. Solid fine lines represent present
day river courses, broken lines palaeo-river courses. The arrows show hypothetical routes of dispersal. The elongate-conic shells represent localities in which
N. burchi is found today, the globose-conic shells indicate the present range of N. aperta. Scale approximate.
reached peninsular Malaysia via the Mekong River that, until the
Pleistocene, had extensions from Cambodia into both peninsular
Malaysia and Borneo (Hall, 1998; Attwood, 2001). The Red River
hypothesis is also supported by the facts that neither Neotricula or
Robertsiella are found in Yunnan, and that an extensive range of
mountains has separated Yunnan from Sichuan (and Hunan)
since the Pliocene; these facts also suggest that Neotricula arrived
in Laos direct from Hunan, rather than via Sichuan-Yunnan
the Pleistocene Mekong River, which ran westwards from central
Laos to northeastern Thailand, and on to the Ping River and
Chiang-Mai (Fig. 3B). Furthermore, both the CO1 and 16S trees
show Robertsiella as the closest relative of Neotricula (out of the taxa
sampled); this also supports the hypothesis of a Hunan origin
for the N. aperta lineage. Although Robertsiella is found only in
Malaysia, its morphologically closest relatives are found in Hunan
(Davis et al., 1992). It is possible that the ancestors of Robertsiella
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S. W. ATTWOOD ET AL.
and the T. bollingi lineage of the Yunnan triculine radiation.
Woodruff, Carpenter, Upatham & Viyanant (1999) suggested that
the ancestors of Oncomelania may have been dispersed over long
distances (and across mountain barriers) on the feet of birds,
presumably trapped in mud. However, this mode of dispersal is
unlikely to have been a factor in the radiation of the Triculinae.
Unlike Oncomelania, triculine snails are not amphibious and do
not survive long out of water.
In summary, the present data have helped substantiate earlier
findings based on morphological characters. Confirmation of
the congeneric status of N. aperta and N. burchi leads to surprising
indications regarding the phylogeography of N. burchi. Although
N. burchi and T. bollingi occur in near sympatry in Chiang-Mai
Province of northern Thailand, the phylogeographies of the two
species are quite different. T. bollingi appears to have evolved
from Yunnanese taxa in the Pliocene Ping– Mekong River system
(the radiation involving only a few hundred km), while the ancestors of N. burchi arose from proto-Neotricula in Hunan and dispersed (and evolved) over thousands of kilometres to northern
Thailand. The findings suggest that current biogeographical
deployment among the Triculinae may not be a good indicator
of phylogeographic relationships. The data have also provided
corroboration for the Robertsiella clade of Hunan by demonstrating the close relationship between Robertsiella and Neotricula.
Together, these conclusions imply that the first major pachydrobiine radiation occurred in Hunan, China. The work described
here is part of an ongoing programme of research aimed at
strengthening phylogeographical hypotheses for Asian Schistosoma through reference to the phylogeographies of their intermediate hosts (see Davis, 1979; Attwood, 2001; Attwood et al.,
2002b). Further work is necessary to obtain more data (molecular
and morphological) for additional triculine taxa, in order to see
if the relationships discussed above remain when new taxa are
added to the phylogenies. It is hoped that through such work a
robust phylogeographical hypothesis for both the Triculinae and
Schistosoma may be obtained.
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ACKNOWLEDGEMENTS
We thank the staff of the Sichuan Institute of Parasitic Disease,
Sichuan Province, for assistance in China, and the field team of
the Institute of Medical Research, Kuala Lumpur. This work was
supported by The Wellcome Trust Project Grant number 058932
and a Royal Society study-visit award to SWA.
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