Lymnaea schirazensis , an Overlooked Snail Distorting Fascioliasis Data: Genotype, Phenotype, Ecology, Worldwide Spread, Susceptibility, Applicability

PMCID: PMC3183092

PMID: 21980347

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Abstract

Background Lymnaeid snails transmit medical and veterinary important trematodiases, mainly fascioliasis. Vector specificity of fasciolid parasites defines disease distribution and characteristics. Different lymnaeid species appear linked to different transmission and epidemiological patterns. Pronounced susceptibility differences to absolute resistance have been described among lymnaeid populations. When assessing disease characteristics in different endemic areas, unexpected results were obtained in studies on lymnaeid susceptibility to Fasciola . We undertook studies to understand this disease transmission heterogeneity. Methodology/Principal Findings A ten-year study in Iran, Egypt, Spain, the Dominican Republic, Mexico, Venezuela, Ecuador and Peru, demonstrated that such heterogeneity is not due to susceptibility differences, but to a hitherto overlooked cryptic species, Lymnaea schirazensis , confused with the main vector Galba truncatula and/or other Galba / Fossaria vectors. Nuclear rDNA and mtDNA sequences and phylogenetic reconstruction highlighted an old evolutionary divergence from other Galba / Fossaria species, and a low intraspecific variability suggesting a recent spread from one geographical source. Morphometry, anatomy and egg cluster analyses allowed for phenotypic differentiation. Selfing, egg laying, and habitat characteristics indicated a migration capacity by passive transport. Studies showed that it is not a vector species (n = 8572 field collected, 20 populations): snail finding and penetration by F. hepatica miracidium occur but never lead to cercarial production (n = 338 experimentally infected). Conclusions/Significance This species has been distorting fasciolid specificity/susceptibility and fascioliasis geographical distribution data. Hence, a large body of literature on G. truncatula should be revised. Its existence has henceforth to be considered in research. Genetic data on livestock, archeology and history along the 10,000-year post-domestication period explain its wide spread from the Neolithic Fertile Crescent. It is an efficient biomarker for the follow-up of livestock movements, a crucial aspect in fascioliasis emergence. It offers an outstanding laboratory model for genetic studies on susceptibility/resistance in F. hepatica /lymnaeid interaction, a field of applied research with disease control perspectives.

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Full Text

Freshwater snails of the family Lymnaeidae (Gastropoda) act as intermediate hosts or vectors of numerous digenean trematode species. Many lymnaeid species are of applied interest as they transmit several trematode species of large-scale medical and veterinary impact, among which fasciolids are the most important [1]. Fasciola hepatica and F. gigantica are two large-sized fasciolid trematode parasite species that cause fascioliasis, a disease which affects humans and livestock species almost everywhere [2]. This highly pathogenic liver parasitosis has been emerging in many countries of Latin America, Europe, Africa and Asia in the last two decades [2], [3]. This emergence phenomenon has partly been related to climate change [4], [5], given the high dependence of both lymnaeid snails and fasciolid larval stages on climatic and environmental characteristics [6]–[8].
Although livestock species play an important reservoir role, transmission studies have shown that the metacercarial infective stage from different origins, such as sheep, cattle, pig and donkey, represent similar infectivity sources [9], [10]. On the contrary, the specificity of fasciolid species regarding concrete lymnaeid species [1] represent a crucial factor in establishing the geographical distribution of the disease in both animals and humans. Moreover, disease prevalences and intensities also depend on the ecological characteristics (population dynamics, anthropophylic characteristics, type of water bodies, etc.) of the different lymnaeid vector species. That is why different lymnaeid species appear linked to the different transmission patterns and epidemiological scenarios of this very heterogeneous disease in humans [11], [12]. The continental differences in lymnaeid faunas also explain that in the Americas fascioliasis is only caused by F. hepatica, owing to the absence of lymnaeids of the genus Radix which act as transmitters of F. gigantica
[1]. Likewise, as in other vector-borne diseases, this relationship supports the use of lymnaeids as biomarkers of the disease at both local and large scales and can thus be useful for the validation of mathematical modelling and remote sensing–geographical information system (RS-GIS) tools for the control of the disease [8], [13].
Despite the applied interest of lymnaeid snails, the present knowledge on the genetics of this gastropod group as well as on their parasite-host interrelationships is far from being sufficient. A good example of this situation is the systematic-taxonomic confusion in which this molluscan family has been immersed [1]. At lymnaeid species level, the problems are found mainly due to the interspecific morphological and anatomic uniformity numerous species present, usually resulting in serious difficulties in specimen classification, sometimes even impeding it [14]–[16]. Moreover, intraspecific variation of shell shape is particularly well marked within lymnaeids depending on environmental conditions [17], [18], although a genetic component in shell shape has been shown at least in some lymnaeid populations [19]. Thus, there are many specimen classification problems, mainly related to: (i) species of the “stagnicoline” group in Europe and North America [14], [20]; (ii) the “radix” group in Europe and Asia [20]; (iii) the “fossarine” or “Galba/Fossaria” group in the Americas [15] (Fossaria is a synonym of Galba
[21]; terms “fossarine” or “Galba/Fossaria” group here used only in the meaning frequently found in American malacological literature of the last century).
At snail host level, trematodes show a marked specificity, from usually oioxenous (one digenean species/one snail species) or stenoxenous (one digenean species/a few, closely related snail species, i.e. those belonging to the same genus) to less frequently oligoxenous (one digenean species/numerous, family-, subfamily- or tribe-related snail species) [22]–[24]. Variability in the susceptibility of a concrete snail species to infection by a concrete digenean species has shown to be related to differences between snail populations and also between individuals among a concrete snail population [24], [25]. Differences in compatibility between a trematode species and different geographical populations of the same snail host species are known, including Fasciola
[26], [27]. Among lymnaeids, pronounced differences in susceptibility have been highlighted among snail populations encountered in close proximity [28], [29], and some snail populations have been mentioned to even show a total lack of susceptibility or resistance [29]–[31].
However, the aforementioned different susceptibility phenomena in lymnaeids have to be considered with great caution. Many of these lymnaeid species involved in fascioliasis transmission maintain a confusing systematic-taxonomic status. When comparing different lymnaeid DNA sequences, several populations originally classified as belonging to different species showed identical DNA marker sequences, and other populations originally classified as pertaining to the same species presented different DNA marker sequences. Sometimes sequence differences were very few, suggesting intraspecific variability (different haplotypes). However, occasionally differences detected among populations classified as pertaining to the same species were numerous, sufficient as to consider different species involved. Moreover, the number of sequence differences between species sometimes appeared lower than that between populations of the same species [1], [14], [16]. This clearly underlines both the classification problems and the systematic-taxonomic confusion present in Lymnaeidae. Consequently, several susceptibility differences described could in fact be related to different lymnaeid species instead of different populations of the same lymnaeid species.
The crucial implications of lymnaeid vectors for fascioliasis transmission, epidemiology and control demonstrate the importance of developing new tools to facilitate specimen classification, genetic characterisation of natural populations and laboratory strains, and to elucidate the systematics and taxonomy of Lymnaeidae. The failure of all malacological and non-malacological tools applied to date suggests the analysis of DNA sequences and phylogenetic methods to be worthwhile. The first attempt made by a research collaboration of parasitologists, molecularists and malacologists was successful [1]. This success, together with the rapid realisation that locally restricted studies were insufficient because of the very large geographical distribution and spreading capacities of many lymnaeids belonging to very confusing lymnaeid groups, suggested early on that large, transboundary studies would be needed. In this sense, a worldwide lymnaeid molecular characterisation initiative was instigated [12], [16]. The great spreading capacity of lymnaeids means that sometimes not even the continental scale is sufficient, and intercontinental sequence comparisons are needed to classify specimens correctly. As an example: the sympatric Lymnaea viatrix and L. cubensis, which were noted to be involved in the human fascioliasis high hyperendemic area of the Bolivian Altiplano [32], later proved to only involve morphologically variable Galba truncatula of European origin [33]–[35]. The intercontinental spreading of lymnaeids and its role in fascioliasis dissemination is well known [2], [27], [36].
Of the different DNA markers used hitherto in lymnaeids, the 18S gene of the nuclear ribosomal DNA (rDNA) appears to be too conserved and its few variable positions may only be useful at generic and suprageneric taxon levels [15], [16], [33], [37]. Ribosomal DNA ITS-2 and secondarily ITS-1 are the most useful sequences for studies at species level [1], [14], [15], [35], [38], [39], [40]. These two spacers are useful for: (i) classification of lymnaeid specimens, (ii) characterisation of lymnaeid intraspecific genetic interpopulational variablity to furnish the genetic base on which to understand fasciolid-lymnaeid specificity, different susceptibilities or compatibilities of geographical strains or even resistances, (iii) establishment of valid species and their geographical distributions, and (iv) assessment of species interrelationships to arrange a natural systematic-taxonomic classification, which will allow for an analysis of coevolution with fasciolids [12], [16]. Interestingly, one mutation at the level of the ITS-1 and another at ITS-2 have proved useful to distinguish between resistant and susceptible populations of P. columella in Cuba [41], although nothing evidently suggests that these mutations are linked to resistance/susceptibility.
Within mitochondrial DNA (mtDNA), only fragments of 16S and cox1 have been sequenced in lymnaeids [15], [40], [42]–[44]. Recent knowledge indicates that mtDNA markers, including both mitochondrial genes and the ribosomal 12S and 16S genes within the mitochondrial genome, should be used with great caution when dealing with lymnaeid species belonging to different genera and even those well-separated within the same genus [45]. Of particular concern is the saturation of nucleotide positions. Additionally, it has also been documented that incomplete gene sequences do not necessarily contain a sufficiently significant portion of the whole gene, i.e. parts of the gene presenting evolutionary hot spots may be missed [46]. Consequently, the use of mtDNA markers for this initiative is restricted to (i) sequence comparisons and phylogenetic analyses of only closely related species within the same genus, (ii) studies of intraspecific variability of species by sequence comparisons of individuals and populations, (iii) genetic characterisation of laboratory strains, (iv) studies on the spread of populations of a species, and (v) studies on genetic exchange between different neighboring populations [12].
The present paper summarises results obtained in analyses of lymnaeid specimens of given populations originally ascribed to the main fascioliasis vector species G. truncatula in Europe, Asia, and Africa, and/or also to similar lymnaeid vector species belonging to the Galba/Fossaria group in North America, the Caribbean and South America. Such analyses were performed after obtaining unexpected results in experimental studies about lymnaeid susceptibility to Fasciola infection when assessing disease transmission characteristics in human hypo- to hyperendemic fascioliasis areas in the different continents. Multidisciplinary studies carried out to understand experimentally-tested, abnormal fasciolid susceptibility of these widely spread lymnaeid G. truncatula-like populations have taken more than 10 years. Characterisation studies have been made by nuclear rDNA and mtDNA sequences, phylogenetic tree reconstruction, phenotypic differentiation by shell morphometry, morphoanatomical characterisation, fecundation studies, ecological observations, and assessment of the geographical spread in correlation with historical events. These results demonstrate that another lymnaeid species, Lymnaea (s. l.) schirazensis, genetically distant but phenotypically very close, has always been confused with G. truncatula and/or other similar lymnaeid vector species in all these continents. The implications for fascioliasis are discussed, as this hitherto overlooked species has been distorting results of fasciolid specificity/susceptibility analyses as well as the geographical distribution of the disease. The existence of this G. truncatula-like lymnaeid species frequently present in animal fascioliasis endemic areas and usual in human fascioliasis endemic areas ought be henceforth considered to avoid misunderstandings concerning transmission. Moreover, results indicate that L. schirazensis can be used as a useful biomarker of foreign livestock introduction, a crucial aspect in fascioliasis spreading and emergence [12]. Additionally, L. schirazensis offers an outstanding new laboratory model for studies on genomics and proteomics about susceptibility/resistance in F. hepatica/lymnaeid interaction, an important field of applied research with disease control perspectives.
The snail specimens studied (n = 8572 specimens) were collected in the field along a ten-year period, from 20 lymnaeid populations present in geographical areas with human and/or animal fascioliasis endemicity of eight countries (Figure 1):
The list of species (and subspecies) of molluscs and trematode parasites included in this study, according to the zoological nomenclature, is included in Table S1. The generic name Lymnaea is used here throughout in the broad taxonomic meaning (sensu lato = s. l.).
DNA was extracted from more than one specimen of a given population when this was deemed necessary for sequence verification. Only snails that appeared free of helminth infection were used in the molecular analyses. To reduce the risk of contamination of DNA from helminths further (which are more likely to be localized in other tissues), DNA was only isolated from the foot of each snail. Use of just the feet, rather than all the soft tissues, also prevented the development in the DNA pellets of the white flocculate substance (probably of polysaccharides) and melanic pigments that can inhibit Polymerase Chain Reaction (PCR) and cause amplification of non-specific products [15], [34].
Snail feet fixed in 70% ethanol were used for DNA extraction procedures. After dissection under a microscope, half of the foot was suspended in 400 µl of lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate SDS) containing 500 µg/ml Proteinase K (Promega, Madison, WI, USA) and digested for 2 hr at 55° C with alternate shaking each 15 min. Steps of the procedure were performed according to methods outlined previously [1], [15], [33]. Total DNA was isolated according to the phenol-chloroform extraction and ethanol precipitation method [47]. Each pellet was dried and resuspended in 30 µl sterile TE buffer (pH 8.0). This suspension was stored at −20° C until use.
DNA sequences were amplified by PCR using 4–6 µl of genomic DNA for each 50 µl PCR reaction, according to methods outlined previously [1], [15], [33], [35]. Each one of the five DNA markers were PCR amplified independently for each lymnaeid specimen and each PCR product was sequenced for a bona-fide haplotype characterization. A set of 8 conserved oligonucleotide primers was used for the amplification of five superimposed fragments of the 18S ribosomal RNA gene using specific primers and a standard protocol [15], [34] to amplify specific 18S rDNA regions. Ribosomal DNA spacers ITS-2 and ITS-1 were amplified using primers designed in conserved positions of 5.8S and 28S rRNA genes and 18S and 5.8S rRNA genes of several eukaryote Metazoa species, respectively [1], [15], [39]. The target 16S gene region was amplified by PCR using a set of universal primers [48]. Amplification procedures and thermal cycler conditions were carried out as previously described for lymnaeids [42]. A cox1 gene fragment was amplified using universal primers [49]. Amplifications were generated in a Mastercycle epgradient (Eppendorf, Hamburg, Germany), by 30 cycles of 30 sec at 94° C, 30 sec at 50° C and 1 min at 72° C, preceded by 30 sec at 94° C and followed by 7 min at 72° C for ITS-2 and ITS-1, and by 40 cycles of 30 sec at 90° C, 1 min at 48° C and 1 min at 72° C, preceded by 2.5 min at 94° C and followed by 10 min at 72° C for cox1. Ten µl of each PCR product was checked by staining with ethidium bromide on 1% Nusieve® GTG agarose (FMC) gel electrophoresis, using Molecular Weight Marker VI (Boehringer Mannheim) at 0.1 µg DNA/µl as control.
The sequencing of the entire 18S rRNA gene, the complete rDNA ITS-2 and ITS-1, and the fragments of the mtDNA 16S and cox1 genes was performed on both strands by the dideoxy chain-termination method [50]. It was carried out with the Taq dye-terminator chemistry kit for ABI 373A and ABI 3700 capillary system (Perkin Elmer, Foster City, CA, USA), using PCR primers.
The codes for the sequences obtained follow the standard nomenclature previously proposed for lymnaeid snails [12], [16], [39]. Note that haplotype codes are only definitive in the case of complete sequences. When dealing with fragments or incomplete sequences, haplotype codes are provisional.
Sequences were aligned using CLUSTAL-W version 1.8 [51] and MEGA 4.0 [52], and assembly was made employing Staden Package [53]. Subsequently, minor corrections were manually introduced for a better fit of nucleotide correspondences in microsatellite sequence regions. Homologies were performed using the BLASTN programme from the National Center for Biotechnology information website (http://www.ncbi.nlm.nih.gov/BLAST). Genetic distances were measured using parameters provided by PAUP v.4.0 b10 [54].
The following sequences from GenBank-EMBL were used for comparative analyses (in the following, names of taxa according to articles in which sequences were described; see Table S1 for systematic-taxonomic notes):
18S rRNA gene: complete sequences of Lymnaea (Lymnaea) stagnalis (GenBank Accession Number Z73984), Lymnaea (Stagnicola) palustris (Z73983), Omphiscola glabra (Z73982), and Galba truncatula (Z73985) [33]; L. cubensis (Z83831) [15], [34], L. viatrix and L. neotropica (both species with the same sequence AM412222) [15], and L. humilis (FN182190) [45], all four from respective type localities; L. cousini and L. meridensis (both species with the same sequence FN598151), and Pseudosuccinea columella (FN598152) [55]; Radix auricularia (Z73980) and R. balthica (Z73981) [33]. Other incomplete sequences available at GenBank were not used to avoid problems in comparative sequence analyses.
rDNA ITS-2: L. (S.) palustris palustris (AJ319620), L. (S.) palustris turricula (AJ319618), L. (S.) fuscus (AJ319621), and Catascopia occulta (AJ319642) [1], [14]; C. catascopium (AF013143), C. emarginata (AF013141, AF013142), C. elodes (AF013138), and Hinkleyia caperata (AF013139) [38]; G. truncatula H1 (AJ296271), H2 (AJ243017), and H3 ( = L. viatrix sensu Ueno et al., 1975; = L. cubensis sensu Ueno et al., 1975) (AJ272051) [1], [15], [35]; L. cubensis (AM412223), L. viatrix (AM412224), L. neotropica (AM412225) [15], L. humilis (FN182191) [45], L. cousini (FN598153), and L. meridensis (FN598154) [55], all six sequences from respective type localities; P. columella (FN598156) [55].
rDNA ITS-1: L. (S.) palustris palustris (AJ626849), L. (S.) palustris turricula (AJ626853), L. (S.) fuscus (AJ626856), and C. occulta (AJ626858) [39]; C. catascopium (AF013143), C. emarginata (AF013142), C. elodes (AF013138), and Hinkleyia caperata (AF013139) [38]; G. truncatula HA (AJ243018), HB (AJ296270), and HC ( = L. viatrix sensu Ueno et al., 1975; = L. cubensis sensu Ueno et al., 1975) (AJ272052) [1], [15], [35], [39]; L. cubensis (AM412226), L. viatrix (AM412227), L. neotropica (AM412228) [15], L. humilis (FN182193) [45], L. cousini (FN598157), and L. meridensis (FN598159) [55], all six sequences from respective type localities; P. columella (FN598160) [55].
mtDNA 16S rRNA gene: Fossaria bulimoides (AF485657), F. obrussa (AF485658), S. elodes (AF485652), and Stagnicola bonnevillensis (AF485655) [43]; F. bulimoides (EU038315) and S. elodes isolate 44106 (EU038305) [56]; L. cubensis (FN182204) and L. humilis (FN182195) [45].
mtDNA cox1 gene: S. elodes (EU038352) [56]; S. elodes (AY227368), F. bulimoides (AY227367), and Austropeplea tomentosa (AY227365) [44]; G. truncatula from Spain (AM494011) [15] and Germany (EU818799) [57]; L. cubensis from Cuba (AM494009–type locality) [15] and the USA (FN182205) [45], L. viatrix (AM494010–type locality), L. neotropica from Peru (AM494008–type locality) [15] and Argentina [58], L. humilis (FN182197-9–type locality) [45], L. cousini (FN598161–type locality, and FN598162-3) and L. meridensis (FN598164–type locality) [55]; P. columella from Australia (AY227366) [44] and Puerto Rico (FN598165) [55].
The previously published secondary structure prediction for Limicolaria kambeul 18S rRNA [59], based on the general eukaryote 18S rRNA secondary structure [60], was used and extended to encompass lymnaeid sequences.
Phylogenies were inferred from DNA sequences using maximum-likelihood (ML) estimates with PAUP [54]. ML parameters such as model, base frequencies, transition/transversion ratio (ts/tv), the shape parameter for the gamma distribution, and the proportion of invariant sites, were optimized using the hierarchical likelihood ratio test (hLRT) and the Akaike information criterion (AIC) [61,62), implemented in Modeltest 3.7 [63]. Starting branch lengths were obtained using the least-squares method with ML distances.
To provide an assessment of the reliability of the nodes in the ML tree, three methods were used. First, a bootstrap analysis using 1000 replicates was made with heuristic search in PAUP. Second, a distance-based phylogeny using the neighbour-joining (NJ) algorithm [64] with ML pairwise distances was obtained. Statistical support for the nodes was evaluated with 1,000 bootstrap replicates, with and without removal of gapped positions. Third, a Bayesian phylogeny reconstruction procedure was applied to obtain posterior probabilities (BPP) for the nodes in the ML tree, by using the same evolutionary model as above, implemented in MrBayes 3.1 [65] with four chains for 1,000,000 generations and trees were sampled every 100 generations. The first 1,000 trees sampled were ruled out (“burn-in”), and clade posterior probabilities (PP) were computed from the remaining trees. Alternative methods of phylogenetic reconstruction allowing for an evaluation of the support for each node were also applied. A distance-based phylogeny using the NJ algorithm with LogDet distances was obtained. Statistical support for the nodes was evaluated with 1,000 bootstrap replicates.
Due to different limitations recently shown by mtDNA markers for interspecific sequence analyses in invertebrates [46], [66], [67], phylogenetic reconstruction by combined sequences data sets was made from ribosomal and mitochondrial markers separately. Combined sets analysed were: a) ITS-1 and ITS-2, considered as the markers to fit best for the analysis of relationships between species, as has already been verified in Lymnaeidae [16]; b) 18S, ITS-1 and ITS-2, to increase the support for evolutionarily older divergence nodes; c) 16S and cox1, to evaluate mtDNA genome information; and d) cox1 gene, by using both the three and only the first two codon positions to assess potential saturation.
Phylogenetic analyses were performed using reference sequences of lymnaeid DNA (see species and Acc. Nos. in list noted above in chapter of sequence comparisons), after adding the following sequences stored at databases: ITS-2: Radix auricularia H1 (AJ319628) and R. balthica H1 (AJ319633); ITS-1: R. auricularia HA (JF922878) and R. balthica HA (JF922879); 16S: L. (L.) stagnalis (AF485659), L. (S.) palustris (U82082), G. truncatula (HQ283236), L. viatrix (HQ283239), L. humilis HA and HB (FN182195, FN182196), L. cousini (HQ283237), L. meridensis HA, P. columella (U82073) and R. auricularia (AF485659). cox1: L. (L.) stagnalis (EU818795), L. (S.) palustris (EU818801), R. auricularia (EU818800). The intergenic region sequence (AY030361) [68] including both ITSs of a planorbid species, Biomphalaria pfeifferi, was used as outgroup. For the combined sets using 18S and 16S, B. alexandrina 18S (BAU65225), ITSs (AY030371) and 16S (AY030204) [68] were used for outgroups, similarly as B. alexandrina cox1 (AF199110).
Shells and egg clusters of lymnaeids were measured, according to traditional malacological methods [19], using a computerised image-analysis system (CIAS) [69]. This system is based on a DXC-930P colour video camera (Sony, Tokyo) fitted to a stereomicroscope, and connected to computer running image analysis software (ImageProH Plus 4.5; Media Cybernetics Inc., Silver Spring, MD).
Egg clusters were obtained from living lymnaeids experimentally maintained in climatic chambers. Living egg clusters were measured with CIAS. For egg clusters, cluster roundness (CR = CP2/4πCA) measurements were used to quantify the cluster shape. It is a measure of how circular an object is (the expected perimeter of a circular object divided by the actual perimeter). A circular object will have a roundness of 1.0, while more irregular objects will have larger values [70].
Isolates of F. hepatica from Poland and Peru, and F. gigantica from Egypt and Vietnam, maintained in the laboratory according to previously described methods [35], were used for experimental infection assays of the laboratory-reared lymnaeids.
Sequence characteristics are noted in Table 1. Data includes length and GC/AT content, obtained for each one of the five DNA markers analysed in the populations of L. schirazensis from the twenty localities of eight countries (Figure 1) with their corresponding haplotype codes and GenBank Accession Nos.
All of the 20 populations of L. schirazensis presented the same 18S sequence (Table 1). When comparing this 18S sequence with that of G. trucatula, a total of 19 variable positions appeared. They included 6 ts, 2 tv and 11 insertions/deletions (indels) in a 1853 bp-long pairwise alingment, most differences being located in the variable Helix E-10 of the V2 region according to the secondary structure (Figure 2).
Helix, Position and Difference number = numbers to be read in vertical. Position = numbers refer to positions obtained in the alignment made with MEGA 4.0. Identical = .; Indel = −. Shaded area corresponds to Helix E10-1 of the variable area V2 where differences in the 18S rRNA gene of Lymnaeidae are concentrated [33]. GenBank Accession Nos. = Z73980−Z73985 [33]; Z83831 [34]; AM4122222 [15]; FN182190 [45]; FN598151- FN598152 [55]; L. schirazensis from present paper. Sequence correspondences: * L. cubensis, L. viatrix, L. humilis and L. cousini without definitive genus ascription; 18S identical in L. viatrix and L. neotropica
[15]; 18S identical in L. cousini and L. meridensis
[55].
A multiple 18S sequence alignment was 1867 bp long. It included: several Galba/Fossaria vector species such as L. cubensis, L. viatrix (the sequence being identical to that of L. neotropica), G. truncatula, L. humilis, and L. cousini (sequence identical to that of L. meridensis); the peculiar species P. columella; three representatives of stagnicolines (L. (L.) stagnalis, L. (S.) palustris and O. glabra); and two of the Radix group (R. auricularia and R. balthica). This alignment showed a total of 64 variable nucleotide positions (3.43% nucleotide divergence). Thirty-two of these 64 polymorphic sites appeared grouped in the short sequence between positions 206 and 266, which corresponds to the helix E10-1 of the variable area V2 of the secondary structure (Figure 2).
Two different ITS-2 haplotypes were found among the L. schirazensis populations analysed (Table 1). Their pairwise comparison showed only 8 polymorphic sites, corresponding to 8 indels caused by the tetranucleotide microsatellite repeat TGCT, present twice in haplotype 1 between positions 128 and 135 of the alignment but absent in haplotype 2.
The very high number of nucleotide differences detected in the pairwise comparisons of these two ITS-2 sequences with the three ITS-2 haplotypes of G. truncatula available at GenBank is noteworthy. In the different alignments performed, with a bp length varying between 469 to 477, a total of 130–139 differences appeared, including 18–19 ts, 11 tv and 101–109 indels (Table 2).
When comparing the L. schirazensis ITS-2 sequences with the other species of the Galba/Fossaria group, the pairwise ITS-2 distance matrix obtained with PAUP (only parsimony informative sites considered) showed that the numbers of nucleotide differences were unexpectedly high in all cases. The lowest of these numbers appeared when comparing with G. truncatula, although rather high (64–66). Regarding L. humilis, L. cousini and L. meridensis, it ranged from 99 to 118. It appeared to be similarly high (121–136) with regard to L. cubensis, L. viatrix and L. neotropica. Nucleotide differences were also numerous when L. schirazensis was compared with stagnicolines and Pseudosuccinea (Table S2).
Two different ITS-1 haplotypes were also found among the L. schirazensis populations analysed (Table 1). The pairwise comparison of these two sequences showed only 3 polymorphic sites, corresponding to two insertions in positions 296 and 297 (AA or –) and to one mutation in position 443 (A or T) in haplotypes A and B, respectively.
A very high number of nucleotide differences also appeared in the pairwise comparisons with the three ITS-1 haplotypes of G. truncatula. In the different alignments performed, with a bp length varying from 558 to 560, a total of 134–138 differences appeared, including 31–32 ts, 21–23 tv and 81–83 indels (Table 2).
When comparing L. schirazensis ITS-1 sequences with the other species of the Galba/Fossaria group, the pairwise ITS-1 distance matrix obtained with PAUP showed that the numbers of nucleotide differences were also very large. The comparison with G. truncatula furnished the second lowest number (81–84), after that of the comparison with L. meridensis (77–80). Regarding L. humilis and L. cousini, it ranged from 87 to 92, and it appeared to be similarly high (83–92) with regard to L. cubensis, L. viatrix and L. neotropica. The number of nucleotide differences increased considerably when L. schirazensis was compared with stagnicolines (132–143) and Pseudosuccinea (129–130) (Table S3).
Two different sequences of the 16S fragment were found (Table 1). They differred by only 2 mutations (C/A in position 85 and A/G in position 305 of their pairwise alignment, in HA/HB, respectively).
Nucleotide comparison with other Galba/Fossaria species showed a total of 46 variable positions (10.75%), of which 31 were mutations (7.24%) and 15 were indels (3.50%). This alignment demonstrated that L. schirazensis was different from any other Galba/Fossaria at the level of this mtDNA gene (Figure 3). In pairwise comparisons, minimum differences were 18 mutations (4.2%) when compared with L. humilis, and a maximum of 22 mutations (5.14%) appeared in comparisn with L. cubensis. Nucleotide differences were more numerous when comparing with the stagnicolines S. elodes and S. bonnevillensis. The four species alignment showed a total of 56 variable positions (12.93%), 47 of which were mutations (10.85%) and 9 indels (2.08%) (alignment not shown).
Numbers (to be read in vertical) refer to positions obtained in the alignment made with MEGA 4.0. Identical = . ; Indel = − . Haplotype codes only provisional due to incomplete sequences of the gene. L. cubensis 16S-HB (FN182204), L. humilis 16S-HA (FN182195) and L. humilis 16S-HB (FN182196) [45]; F. bulimoides (AF485657) and F. obrussa (AF485658) [43]; * the sequence ascribed to the stagnicoline C. elodes (EU038305) [56] concerns in fact a Galba/Fossaria species-see analysis in [45].
Four different cox1 sequences were obtained (Table 1). When aligned, a total of 661 positions were conserved and only 11 were variable, comprising 2 parsimony informative and 9 singleton sites (Table 3). When comparing with species of the Galba/Fossaria group and other proximal lymnaeid species available at GenBank, whose cox1 fragment sequences were similar in length to those obtained in the present study, the higher number of nucleotide differences appeared evident in a pairwise cox1 distance matrix (Table S4). A comparison with more distant species such as stagnicolines (S. elodes) and Radix group (Austropeplea tomentosa) was not necessary.
The amino-acid sequence of that COX1 gene fragment was 224 aa long. A pairwise comparison showed a 100% identity between the three L. schirazensis haplotypes cox1-a, cox1-b and cox1-c, and only one amino-acid difference (isoleucine/threonine, respectively) in position 96 with L. schirazensis haplotype cox1-d from Baños del Inca, Peru. The provisional haplotypes COX1-I and COX1-II were assigned, respectively. The comparison with the amino acid sequence of other species of the Galba/Fossaria group and other proximal lymnaeid species showed that COX1-I of L. schirazensis was identical to that of other species, such as L. viatrix, L. humilis and L. meridensis. However, L. schirazensis COX1-II appeared to be unique (Figure 4).
This single data-set generated a robust tree, indicating phylogenetic accordance between the two spacers. The ML model best fitting this data-set was HKY85+I, using a ts/tv ratio of 2 (kappa = 3.8979086), base frequencies for A, C, G and T of 0.21534, 0.26862, 0.22661 and 0.28943, respectively, and a proportion of invariable sites = 0.087. To assess the reliability of the nodes in the ML tree (Figure 5 A), a bootstrap analysis using 1,000 replicates was made using branch-swapping algorithm (tree-bisection-reconnection TBR) with heuristic search and the neighbour-joining (NJ) algorithm with the ML pairwise distances in PAUP. Finally, a Bayesian phylogeny reconstruction procedure was applied to obtain posterior probabilities (BPP) for the nodes in the ML tree with MrBayes 3.1.
Scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; b: bootstrap with ML reconstruction using PAUP with 1000 heuristic replicates; c: Bayesian posterior probability with ML model using MrBayes. See Table S1 for systematic-taxonomic notes.
In the ML tree obtained (Figure 5 A), P. columella appeared basal to two large groupings, namely stagnicolines (including both Palaearctic and Nearctic species), and the Galba/Fossaria clade which comprised the F. hepatica main vector species. Lymnaea schirazensis appeared included within the latter. However, contrary to what would be expected, L. schirazensis did not cluster together with G. truncatula, of Old World origin, or other morphologically similar lymnaeids such as the New World L. cubensis, L. viatrix and L. neotropica. Surprisingly, it appeared linked to the Nearctic L. humilis and the two Neotropical L. cousini and L. meridensis, within a relatively well supported branch (82/78/88 in NJ/ML/BBP).
The topology obtained with the NJ algorithm using LogDet distances (figure not shown) was identical to that shown by the ML tree (Figure 5 A).
When adding two Radix species (data matrix of 24 taxa with 1,767 characters), the ML model best fitting was HKY85+G+I, using a ts/tv ratio of 1.45 (kappa = 2.872483), base frequencies for A, C, G and T of 0.22764, 0.25354, 0.23176 and 0.28705, respectively, a shape parameter (alpha) = 0.99, and a proportion of invariable sites = 0.13. In the ML tree obtained (Figure 5 B), Radix species clustered independently in a branch basal to all the remaining lymnaeids. Unexpectedly, the location of L. schirazensis changed, now becoming basal to the other Galba/Fossaria members which clustered together although with very low supports. The topology of the NJ LogDet tree was similar (figure not shown), but with L. schirazensis in a subclade with G. truncatula showing low bootstrap (only 59).
This three ribosomal marker data set (matrix of 17 taxa and 3,540 characters) also generated a robust tree. The ML model best fitting was HKY85+I, using a ts/tv ratio of 1.48 (kappa = 2.9601982), base frequencies for A, C, G and T of 0.22150, 0.25100, 0.25820 and 0.26930, respectively, and a proportion of invariable sites = 0.44. In the ML tree obtained (Figure 6 A), L. schirazensis appeared clustering together with G. truncatula (79/81/- in NJ/ML/BPP), and the branch including L. humilis, L. cousini and L. meridensis appeared as the sister group inside the Galba/Fossaria clade. The topology obtained with the NJ algorithm using LogDet distances (figure not shown) was identical.
Scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; b: bootstrap with ML reconstruction using PAUP with 1000 heuristic replicates; c: Bayesian posterior probability with ML model using MrBayes. See Table S1 for systematic-taxonomic notes.
The data matrix analysed contained 19 taxa and 1,118 characters. The ML model best fitting was HKY85+G+I, using a ts/tv ratio of 1.78 (kappa = 5.083228), base frequencies for A, C, G and T of 0.33500, 0.10560, 0.12210 and 0.43730, respectively, a shape parameter (alpha) = 0.39, and a proportion of invariable sites = 0.35. The ML tree (Figure 6 B) showed L. schirazensis haplotypes clustering together with the branch of L. humilis, L. cousini and L. meridensis, although with low support values (60/–/60). The topology furnished by the NJ algorithm using LogDet distances (figure not shown) was similar, with that clade supported by a 64% bootstrap.
Shiraz, Iran [71].
The shell was brownish to reddish light brown, thin-walled, elongated conical, usually with four regular convex whorls and up to 5.5 whorls in the longer specimens (Figure 7). The whorls were somewhat inflated, slightly shouldered, with silky and longitudinally striated surface and separated by a deep, well-marked suture, increasing rather slowly in diameter. The columella was straight, unfolded, and the umbilicus open. The last or body whorl was almost ¾ times as high as the shell height, presenting a slight twisting trend along its basal part visible in the biggest shells when viewed dorsally, and which was due to the enlargement of the basal lip of the peristome (in the way of Pseudosuccinea columella). The spire was pointed. The aperture was elongatedly oval, slightly oblique, mid-sized and wider at the base. The thin peristome was patent throughout, the umbilicus partially covered by a widened columellar lip. The shell showed a tendency to be approximately one and a half to two times as long as it is wide, and its aperture tends to be slightly less than half as long as the shell. Measurements and calculated ratios of natural and experimental populations were noted (see Table 4 and Table S5, respectively).
The cephalopedeal mass was pale greyish. The eyes were black, relatively big in size (Figure 8 A–C). Tentacles were elongate, slender, pyramidal, with narrow base (Figure 8 A–E). The mantle roof was dark, from dark brown to blackish throughout, with small unpigmented white-greyish round spots including several tiny circles at the beginning of the border of the pulmonary region and a few scattered further away in between the initial large round spots (Figure 8 F–I). The border of the mantle was light grey. The black pigmentation of the hypopeplear region of the mantle roof gave a dark appearance to the shell of living specimens by transparency (Figure 8 D,E). This dark appearance did not depend on the characteristics of the natural habitat, as it was maintained across the different laboratory-reared snail generations (quite the opposite of what happens with several darkish populations of other lymnaeid species under experimental conditions).
Morphoanatomical features were studied (see Figure 9 and Figure S1). The renal tube extended straight from the pericardial region toward the mantle collar, diagonally across the roof of the palial cavity and parallel to the pulmonary vein and renal vein. It was a straight tube, lined with a folded epithelium, white to white-yellowish, that tapered, smoothed and became transparent distalward until the level of the septum between pulmonary and hypopeplear cavities. In that place, it turned with an almost right-angle to form a short ureter (which lacked the two distinct flexures present in other close species) and finally opened behind the pneumostome. A very slender muscle thread appeared lengthwise on the ventral surface of the renal tube. A layer of very thin transverse parallel muscle fibers gave the inner side of the pulmonary wall a densely striated appearance (Figure 9 A,B).
The ovotestis showed a lobulate surface formed by several acini, with a collecting canal which continued into the ovispermiduct. The latter presented a short and very thin proximal segment emptying into the seminal vesicle, an expanded portion of bosselated surface, which narrowed down into a slender distal segment ending in the carrefour surrounded by the albumen gland, oviduct and spermiduct (Figure 9 C–F).
The voluminous albumen gland covered the carrefour and the origin of a bosselated, transverse tubular oviduct which followed a somewhat convolute course, described a nearly complete circle in contact with the albumen gland, and continued into a striated, oblong nidamental gland (Figure 9 C,D and Figure S1 A,B). The initial part of the nidamental gland increased in width to keep the diameter throughout its length or even slightly enlarged in its distal part, to subsequently narrow to give rise to a wrinkled-walled uterus followed by a short vagina. The latter shows a first conical narrowing part and a final short tubular part (without any bulbous swelling or sphincter-like thickening present in other close species) (Figure 9 E–G and Figure S1 A,B), opening into the female genital pore with a slightly thickened lip. All parts of the female reproductive system proximal to the vagina itself showed a pronounced width.
The yellowish spermatheca (Figure S1 A,B) had an oval body and gave rise to a thin, relatively long spermathecal duct which emerged laterally from the spermatheca instead of from its terminal extremity. The almost uniformly thin, smooth-walled spermathecal duct extended diagonally between the nidamental gland and the prostate until joining the final part of the uterus and the beginning of the vagina. The spermathecal duct was not dilated at its distal end (Figure 9 E–G and Figure S1 B).
Emerging from the carrefour, the spermiduct showed an initial slightly dilated part with a granular outer surface slendering into a uniformly thin, straight duct which ran on the ventral surface of the nidamental gland. The distal portion of the spermiduct widened to form a prostate of granular surface, relatively small size, narrower than the nidamental gland. The small, oblong, light grey prostate gland varied in shape from inverted-pear-like to elongated-potato-like, without ventral lengthwise fissure (Figure 9 E,F and Figure S1 C). In cross section, the prostate showed a simple, slit-like lumen (lacking internal folds known in other lymnaeids) (Figure 9 H and Figure S1 D). The prostate gave distally rise to a smooth-walled vas deferens which ran shortly in contact with the vagina and extended until looping caudalward. After this loop, the vas deferens became a uniformly thin, long duct to finally merge into a curved, penial sheath or phallotheca (Figure 9 E,F and Figure S1 E,F).
The penis sheath was regularly cylindrical, with a very slightly thicker proximal part (which showed no patent circlet of minute knobs or ring of papillae described in close species). The penis sheath was about a little bit more than half as long than the praeputium. The maximum width of the praeputium was around two or even three times thicker than the penis sheath. The shape of the praeputium was usually elongate-conical, gradually narrowing to terminate in the male genital pore, although it also appeared cylindrical thoughout most of its length and only tapering distally (Figure 9 E,F and Figure S1 E,F). This conical shape of the praeputium appeared to be an exception among the typical conical praeputium shape in Lymnaeidae. The length of the praeputium measured 0.73–1.96 mm (mean 1.13 mm) while that of the penis sheath was 0.36–0.94 mm (mean 0.72 mm), with a praeputium length/penis sheath length ratio of 1.20–2.23 (mean 1.60).
Shape variability of egg clusters and eggs was studied (Figure 10), and measurements and calculated ratios were noted (Table 5). Egg clusters were saccular, transparent, with a very thin outer membranous wall. The shape of these egg clusters showed an evident trend from kidney- to banana-like; the more curved and relatively more elongated and narrow the more numerous were the eggs inside. Small clusters containing only very few eggs (2–5) appeared rounded to oval, but the elongate-curved shape became soon apparent when the egg number increased. Eggs were from spherical to slightly ovoid, with a uniformly thick outer shell, usually appearing well separated from each other.
In nature, this species was almost never found in water, nor even close to water edges, sometimes up to 1 m or farther from it (Figure 11 I). Outside water, it was usually found on the soil surface under grass besides slow running large rivers (Figure 11 B) or even under relative high bushes completely away from sunhine (Figure 11 H). The smallest amount of water to keep humidity was sufficient for this species to maintain populations, as in ditches of rural paths and dirt tracks (Figure 11 L,N) or even artificial cement canals in gardens (Figure 11 A) and drainage canalizations around buildings and dwellings (Figure 11 K). Man-made small irrigation canals around plant cultures also appeared to offer appropriate conditions for its development (Figure 11 C,D), as well as large drainage cement canals with almost no vegetation (Figure 11 E). Worth mentioning was its presence on mud and livestock footprints in and around animal farms (Figure 11 M).
Besides this link to antropophilic habitats and livestock-inhabited places, it was also found, however, in more silvatic habitats. Examples were specimens under the dense vegetation of the edges of fast running waters in mountain rivers and streams, where populations undoubtedly derived from dragging and floods by the water from upper areas (Figure 11 F,J,G).
Worth mentioning was that specimens of L. schirazensis sometimes appeared so mixed or close to one another with specimens of G. truncatula, that one was convinced to deal with a population of only one species, e.g. in Spain (Figure 11 D,E).
Egg cluster laying was verified to occur in all of the specimens from different populations maintained individually isolated after hatching (Table 6). Age and size when laying began differed somewhat from one individual to another within a population, less in populations of the New World (Mexico) than in those of the Old World (Egypt, Spain). Despite range overlap, those from Mexico proved to be markedly sexually precocious in age when compared to those from Egypt and Spain. Snail size when laying began did not show a similar correlation, suggesting that development speed and reaching a sufficient size may be more important for sexual activity.
Comparison of the measurements of the first and first five clusters laid by each individual snail also showed that slight differences may be found according to the populations. Results indicated that there is no gradual increase in cluster size nor in the number of eggs per cluster within the laying of a snail specimen. Thus, sometimes the first egg cluster laid by a snail individual was larger and contained more eggs than those laid afterwards (2nd to fifth clusters) by the same specimen (Table 6). However, somewhat larger clusters including a slightly larger number of eggs/cluster were laid by older and larger snail specimens (up to 20 and 22 eggs/cluster in specimens from Egypt and Spain, respectively).
Snail life span was relatively short, of around 2.5–6.8 months. It includes a sexually active period of cluster laying lasting a total of 39–161 days, sometimes even only very few days before death. However, the final postlaying period may reach slightly more than 1.5 months in given specimens (Table 6 and Table 7; Figure 12). Both life span and laying period appeared to be longer in Old World (Egypt, Spain) specimens than in the New World (Mexico) specimens.
Populations studied: Albufera of Valencia, Valencia, Spain; Tiba, Delengate district, Egypt; Escuela A. Obrego, La Trinidad Tepango, Atlixco, Puebla, Mexico; and Jiutepec, Morelos, Huauchinango, Mexico. n = 10 snail specimens followed per population (for details on intrapopulational variability ranges during the laying period, see Table 6 and Table 7); tlc = mean total laying capacity (number of clusters/life span in days); lrsp = mean laying rate in sexually active period (number of clusters/laying period in days); tnld = mean total non-laying days within laying period.
Usually, snails only laid one egg cluster on the days they laid. Two clusters were sometimes laid by a snail specimen in the same day, although this appeared to be very rare in Old World (Egypt, Spain) specimens and occurred almost never (only once) in the New World (Mexico) specimens. Throughout the laying period, the number of days when no cluster was laid was larger in Old World (Egypt, Spain) specimens than in New World (Mexico) specimens. This fact seemed to be mainly related to the longer laying period in Old World specimens, as the differences in the maximum number of non-laying days between two laying days when comparing Old and New World specimens were too small (Table 7; Figure 12).
Several experimental infection assays including a total of 338 snail specimens from different geographical origins with miracidia from experimentally-reared strains of F. hepatica and F. gigantica, also from different geographical origins, did not allow for successful fluke larval development. None of the infected specimens appeared to become parasitised nor reached cercarial shedding (Table 8). Only in two specimens from Albufera of Valencia, Spain, infected with miracidia of F. hepatica from Poland, could a few rediae be observed by transparency through the snail shell at 23 dpi, although neither shed cercariae thereafter. In a subsequent experiment with the same snail- and fluke-strains for verification, no infection and not even rediae were obtained in spite of the higher number of lymnaeid specimens infected.
Results on survivorship of snails after infection showed pronounced differences according to snail strains and fluke species and strains used. There were experiments in which no snail infected by either F. hepatica or F. gigantica was able to survive after 30 dpi, up to other experiments in which snails survived for more than 100 or even 150 dpi, thus suggesting no negative effects. Nonetheless, results suggested a lower survival rate in infections by F. gigantica than by F. hepatica. Furthermore, in F. hepatica infection assays, a faster and higher snail mortality rate appeared when using trimiracidial instead of monomiracidial infection (Table 8).
The twenty lymnaeid populations found in the eight countries of Asia, Africa, Europe, the Caribbean, North America and South America, show DNA sequences proving that all of them belong to the same species. The fact that this species presents an 18S rRNA sequence only is easily understandable given the slow evolution characteristics of this highly conserved gene [33], [34]. The low number of nucleotide differences at rDNA ITS-2 and ITS-1 level fits within the known intraspecific variability ranges of these two species markers [1], . However, it poses a question mark when considering the very wide geographical distribution (Figure 1), markedly variable habitats (Figure 11), and ecology (e.g., large altitudinal range) this lymnaeid species shows. Even the low number of only two and four haplotypes in the mtDNA 16S and cox1 gene fragments, respectively, including but a very few variable positions in both genes, is also surprising given the faster evolution of these mtDNA genes when compared to nuclear rDNA genes and spacers in invertebrates in general [46] and in lymnaeids in particular [45].
The interspecific comparison of the sequences of the snail species here in question demonstrates that it is a unique lymnaeid. It is genetically well different from G. truncatula and other species of the Galba/Fossaria group, as well as from stagnicolines and P. columella. At 18S rRNA level, L. schirazensis presents peculiar nucleotides in the helix E10−1 of the variable V2 area, which clearly differs from all other species (Figure 2). This fact in a well conserved gene such as 18S is worth mentioning [34]. At ITS-2 and ITS-1 level, the two main markers for species distinction [1], [16], [39], the very high number of nucleotide differences L. schirazensis shows when compared to all other lymnaeids must be highlighted. This is surprising when considering the pronounced morphological similarity of L. schirazensis with other lymnaeid species, mainly of the Galba/Fossaria group.
Concerning the sequences of the 16S and cox1 gene fragments, nucleotide differences between L. schirazensis and all other lymnaeids are also numerous (Figure 3 and Table S4), although less pronounced than those appearing in both ITSs despite the faster evolution of mtDNA. This may be related to the well known saturation of nucleotide positions in these two mitochondrial genes, a phenomenon previously described in Lymnaeidae [45].

Galba truncatula is the lymnaeid species on which the largest number of studies has focused for decades, and one of the snail species with the largest body of literature and longer list of synonyms among molluscs in general. This very wide multidisciplinary body of literature is related to its applied importance as the main vector of F. hepatica throughout many countries [12], [29], in a life cycle which was the first to be elucidated among trematodes [72]. Thus, it is surprising that another widely distributed lymnaeid species, similar in morphology, biology and ecology, has always been confused with and kept masked under G. truncatula until now.
The very long list of species and varieties described long ago and later synonymised with G. truncatula
[73] has been reviewed to analyse whether a species description published in the past could fit the characteristics of the lymnaeid species here in question. The older species name, fully fitting our material, appears to be Lymnaea schirazensis, described in 1862–1863 [71] from material collected in the locality of Shiraz, Iran, by von dem Busch who never published the description of his snail material (see Figure S2 and Figure S3). Consequently, the decision has been taken to ascribe the material described herein to the binomium Lymnaea schirazensis Küster, 1863. This species cannot be found in the literature published in recent decades. This ‘disappearance’ was undoubtedly due to its synonymy with G. truncatula, a proposal of Hubendick [73] apparently accepted by other specialists. Lymnaea schirazensis appears to have been given systematic validity and reported separately from G. truncatula only in Russia [74].
An exhaustive analysis of the old literature (see numerous references in [73]) suggests that several old species and variety names also synonymised with G. truncatula probably referred to L. schirazensis instead of to G. truncatula. The following items, in chronological order, are worth listing given the geographical locality the type materials came from:
the four G. truncatula varieties distinguished in Algeria [75] suggest that at least one of them could be the species here in question: major (not that of Moquin-Tandon) from Mostaghanem, minutissima from Alger, Géryville and Bône, submalleata from Djelfa, and lanceolata from Alger;

L. persica from the same type locality of Shiraz, Iran [76] is a synonym already previously proposed [77];

L. hordeum from River Euphrates, Mesopotamia [78];

L. delaunayi from Pasajes, northern Spain [79];
the variety thiesseae from Euboea, Greece [80] is very probably synonymous; this synonymy was already aptly discussed [81];
the two varieties neapolitana from Sebeto, near Naples, Italy [82] and telouetensis from Mount Atlas [83] merit further analysis.
The insufficient original description of L. zrmanjae from Zrmanja river, Dalmatian Croatia [84] may also lead to suspect a synonymy, although erroneously, as the latter indeed belongs to the family Hydrobiidae, as verified by the same author somewhat later (Brusina, 1902 in [85]) (see also Table S1).
The coexistence with other very similar lymnaeid species of the Galba/Fossaria group within the same fascioliasis endemic areas, such as G. truncatula in the Old World, and L. cubensis, L. humilis, L. cousini and L. neotropica additionally to G. truncatula in the New World, suggests that a wider distribution of L. schirazensis may have been masked by lymnaeid specimen misclassifications. All of the aforementioned lymnaeids are vectors of fascioliasis and very easily confused when only classified from external characteristics or by non-expert scientists, mainly when specimens are small or mid-sized [15]. A size larger than the one of L. schirazensis is reached by Galba truncatula
[29], L. cubensis
[86], L. humilis
[87], and L. cousini
[88], [89]. Only L. neotropica is of a size similar to that of L. schirazensis
[15]. Galba truncatula is present in the Gilan province of Iran [89], in the Nile Delta region [90], [91], along the Mediterranean coast of the Valencian Community, Spain [1], [92], and in Merida State of Venezuela [93]. Lymnaea cubensis is known throughout Hispaniola, including both the Dominican Republic [94] and Haiti [95], Puebla province of Mexico [96] and Merida State of Venezuela [93], [97]. Lymnaea humilis is the species most frequently mentioned to be involved in fascioliasis transmission in Mexico (see review in [45]), sometimes under the names of L. modicella and L. obrussa
[28], [98], both synonymised with L. humilis
[73], and also reported in Puebla State [96]. Lymnaea cousini has been reported in fascioliasis endemic Andean areas of Ecuador [55], [73], [99], and also in Merida State of Venezuela [88], [93]. However, this Venezuelan material later proved to belong to another species, namely L. meridensis
[55]. Lymnaea neotropica has been reported, under the name of L. viatrix
[15], in several fascioliasis endemic Andean valleys of Peru, as is the case of Cajamarca [100], [101].

L. schirazensis: specific classification can be based on the nucleotide sequences of ITS-2 rDNA, ITS-1 rDNA, mtDNA 16S, and mtDNA cox1; for supraspecific classification, the nucleotide sequences of the 18S rDNA and of both ITSs can be used (Table 1); the amino-acid sequence of the mtDNA COX1 protein does not appear to be helpful for any discrimination.

G. truncatula: specific classification can be based on the nucleotide sequences of ITS-2 rDNA [1], [15], [35]; ITS-1 rDNA [15], [35], [39], and mtDNA cox1 [15], [57]; for supraspecific classification, the nucleotide sequences of the 18S rDNA [33] and of both ITSs can be used; the mtDNA COX1 amino-acid sequences [15], [57] do not appear to be useful at any level.
Shell size: smaller in L. schirazensis (Table 4), reaching a shell length of up to 12.00 mm in G. truncatula
[29], whereas it shows a maximum of only 8.06 mm in L. schirazensis; other shell characteristics may be helpful, such as the regularly convex whorls and straight columella in L. schirazensis (Figure 7 A–D) when compared to the stepped whorls and folded columella in G. truncatula (Figure 13 A–F); however, shell variability may easily give rise to confusion when based only on these aspects;

Tentacles: of different form, being elongate, slender and with a narrow base in L. schirazensis (Figure 8 A–E), whereas they are wider and with a wide base in G. truncatula (Figure 13 G);
Eyes: of different size, being relatively big and markedly larger in L. schirazensis than in G. truncatula (Figure 13 H,I);
Egg clusters: of different shape trend, showing an evident trend from kidney- to banana-like, the more curved, elongated and narrow the more numerous are the eggs inside, in L. schirazensis (Figure 10 A–O); clusters tend to keep a rounded to oval shape even when containing more eggs, in G. truncatula (Figure 10 P–Z);
Egg number per cluster: although no significant differences were found between both species in our study, according to the literature it appears that a higher mean egg number per cluster of 12–15 may be the usual rule in G. truncatula, with exceptions of a lower number depending on the soils of the different areas and sometimes even on the season [29]; this mean number shows a lower level range of around 6–14 in L. schirazensis (Table 5);
Mantle colour: mantle roof from dark brown to blackish throughout, with unpigmented white-greyish round spots, this black pigmentation giving a dark appearance to the shell of living specimens by transparency in L. schirazensis (Figure 8 D–I); in G. truncatula the hypopeplear region of the mantle roof shows larger unpigmented whitish spots giving a pale appearance to the shell of living specimens by transparency (Figure 13 I–K);
Male organs: with different praeputium length/penis sheath length ratio, which is of 1.20–2.23 (mean 1.60) in L. schirazensis (Figure 9 E,F and Figure S1 E,F); in G. truncatula the length of the praeputium is 0.82–2.80 mm (mean 1.41 mm) and that of the penis sheath is 0.22–0.64 mm (mean 0.37 mm), with a praeputium length/penis sheath length ratio of 2.50–5.90 (mean 3.44), according to data obtained by measuring specimens from Öland, (Sweden), Kühren, Ettenheim and Reusten (Germany), and Scharflinge (Austria) (Figure 13 M,N);
Ecology: with different water dependence, showing marked amphibious characteristics with a pronounced terrestrial behaviour in L. schirazensis (see habitat types in Figure 11 A–M), whereas aquatic inside freshwater borders or amphibious but more water-dependent in G. truncatula.
The phylogenetic analyses performed (Figure 5 A,B and Figure 6 A,B) confirms the ascription of L. schirazensis to the Galba/Fossaria group, as already suggested by the morphoanatomic characteristics of this species. Thus, its transfer to the genus Radix as proposed by some researchers (e.g., http://clade.ansp.org/malacology/collections/search.php?mode=browsetypes&targetfamily=Lymnaeinae) does not appear supported.
Unfortunately, there is great confusion around the generic names proposed for truncatula and proximal species in the Americas (the Central American cubensis, the North American humilis and bulimoides, and the South American viatrix and diaphana, only to mention the most important), all of which share the capacity of transmitting F. hepatica
[1]. Besides the genus Galba Schrank, 1803 (type species: truncatula) [1], [14], [20], [34], [102], [103], in which it was included [50], other genera having frequently been used to include the New World Galba/Fossaria species, such as cubensis and viatrix, are: Fossaria Westerlund, 1885 (type species: truncatula) [104]–[107], Nasonia Baker, 1928 (type species: cubensis) (see [73]), the subgenus Fossaria (Bakerilymnaea) Weyrauch, 1964 (type species: cubensis) [104], [106], [108], and the subgenus Lymnaea (Afrogalba) Kruglov et Starobogatov, 1985 (type species: misassigned to cubensis; correct type species is mweruensis Connolly, 1929) [109].
In the same clade with L. humilis, L. cousini and L. meridensis: That situation was the one obtaining higher support values, appearing in a higher number of phylogenetic trees, and supported by both rDNA (ITSs) and mtDNA (16S−cox1) sets. This may support the classification of L. schirazensis within the genus Pseudogalba Baker, 1913 (new name proposed for Simpsonia Baker, 1911; type species: humilis) (see [73]). However, the node branching L. schirazensis together with L. humilis, L. cousini and L. meridensis never appeared with sufficiently high support values.
Ecological characteristics of L. schirazensis are peculiar when compared to other Lymnaeidae. The amphibious behaviour differ between lymnaeid groups. Radix and stagnicoline species prefer permanent, larger and deeper water bodies, which they leave only sporadically. On the opposite, Galba/Fossaria species may be found on mud or wet sites very close to water in both temporary and permanent water bodies [1], [5]. This approaches L. schirazensis to Galba/Fossaria. However, no other lymnaeid shows such a preferential terrestrial behaviour.
Its amphibious characteristic and wide habitat range, mainly its presence in habitats with very small amounts of freshwater, suggest a great adaptation capacity. Additionally, its frequency in antropophilic and man-made habitats and its survival capacity outside water suggests that this snail can be passively transported by livestock and/or human activities, as demonstrated in G. truncatula
[12], which shows similar habitat characteristics [29].
Selfing in L. schirazensis remembers the autofecundation preference in G. truncatula
[29], [110]–[113] and other Galba/Fossaria species (Khoubbane et al., unpublished). Its laying capacity (Table 6 and Table 7) appears to be greater than in G. truncatula
[29], with a higher number of clusters/month laid per specimen along the laying period. Both selfing and high laying capacity will undoubtedly facilitate its capacity to colonise neighbour

---

Sections

"[{\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues1\", \"pone.0024567-MasComa1\", \"pone.0024567-MasComa1\", \"pone.0024567-World1\", \"pone.0024567-MasComa2\", \"pone.0024567-MasComa3\", \"pone.0024567-Ollerenshaw1\", \"pone.0024567-Fuentes1\"], \"section\": \"Introduction\", \"text\": \"Freshwater snails of the family Lymnaeidae (Gastropoda) act as intermediate hosts or vectors of numerous digenean trematode species. Many lymnaeid species are of applied interest as they transmit several trematode species of large-scale medical and veterinary impact, among which fasciolids are the most important [1]. Fasciola hepatica and F. gigantica are two large-sized fasciolid trematode parasite species that cause fascioliasis, a disease which affects humans and livestock species almost everywhere [2]. This highly pathogenic liver parasitosis has been emerging in many countries of Latin America, Europe, Africa and Asia in the last two decades [2], [3]. This emergence phenomenon has partly been related to climate change [4], [5], given the high dependence of both lymnaeid snails and fasciolid larval stages on climatic and environmental characteristics [6]\\u2013[8].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Valero1\", \"pone.0024567-Valero2\", \"pone.0024567-Bargues1\", \"pone.0024567-MasComa4\", \"pone.0024567-MasComa5\", \"pone.0024567-Bargues1\", \"pone.0024567-Fuentes1\", \"pone.0024567-Fuentes2\"], \"section\": \"Introduction\", \"text\": \"Although livestock species play an important reservoir role, transmission studies have shown that the metacercarial infective stage from different origins, such as sheep, cattle, pig and donkey, represent similar infectivity sources [9], [10]. On the contrary, the specificity of fasciolid species regarding concrete lymnaeid species [1] represent a crucial factor in establishing the geographical distribution of the disease in both animals and humans. Moreover, disease prevalences and intensities also depend on the ecological characteristics (population dynamics, anthropophylic characteristics, type of water bodies, etc.) of the different lymnaeid vector species. That is why different lymnaeid species appear linked to the different transmission patterns and epidemiological scenarios of this very heterogeneous disease in humans [11], [12]. The continental differences in lymnaeid faunas also explain that in the Americas fascioliasis is only caused by F. hepatica, owing to the absence of lymnaeids of the genus Radix which act as transmitters of F. gigantica\\n[1]. Likewise, as in other vector-borne diseases, this relationship supports the use of lymnaeids as biomarkers of the disease at both local and large scales and can thus be useful for the validation of mathematical modelling and remote sensing\\u2013geographical information system (RS-GIS) tools for the control of the disease [8], [13].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues1\", \"pone.0024567-Bargues2\", \"pone.0024567-Bargues4\", \"pone.0024567-Burch1\", \"pone.0024567-Burch2\", \"pone.0024567-Samadi1\", \"pone.0024567-Bargues2\", \"pone.0024567-Gler1\", \"pone.0024567-Gler1\", \"pone.0024567-Bargues3\", \"pone.0024567-International1\"], \"section\": \"Introduction\", \"text\": \"Despite the applied interest of lymnaeid snails, the present knowledge on the genetics of this gastropod group as well as on their parasite-host interrelationships is far from being sufficient. A good example of this situation is the systematic-taxonomic confusion in which this molluscan family has been immersed [1]. At lymnaeid species level, the problems are found mainly due to the interspecific morphological and anatomic uniformity numerous species present, usually resulting in serious difficulties in specimen classification, sometimes even impeding it [14]\\u2013[16]. Moreover, intraspecific variation of shell shape is particularly well marked within lymnaeids depending on environmental conditions [17], [18], although a genetic component in shell shape has been shown at least in some lymnaeid populations [19]. Thus, there are many specimen classification problems, mainly related to: (i) species of the \\u201cstagnicoline\\u201d group in Europe and North America [14], [20]; (ii) the \\u201cradix\\u201d group in Europe and Asia [20]; (iii) the \\u201cfossarine\\u201d or \\u201cGalba/Fossaria\\u201d group in the Americas [15] (Fossaria is a synonym of Galba\\n[21]; terms \\u201cfossarine\\u201d or \\u201cGalba/Fossaria\\u201d group here used only in the meaning frequently found in American malacological literature of the last century).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Wright1\", \"pone.0024567-Adema1\", \"pone.0024567-Adema1\", \"pone.0024567-Rollinson1\", \"pone.0024567-Boray1\", \"pone.0024567-Boray2\", \"pone.0024567-PerezReyes1\", \"pone.0024567-Rondelaud1\", \"pone.0024567-Rondelaud1\", \"pone.0024567-Gutierez1\"], \"section\": \"Introduction\", \"text\": \"At snail host level, trematodes show a marked specificity, from usually oioxenous (one digenean species/one snail species) or stenoxenous (one digenean species/a few, closely related snail species, i.e. those belonging to the same genus) to less frequently oligoxenous (one digenean species/numerous, family-, subfamily- or tribe-related snail species) [22]\\u2013[24]. Variability in the susceptibility of a concrete snail species to infection by a concrete digenean species has shown to be related to differences between snail populations and also between individuals among a concrete snail population [24], [25]. Differences in compatibility between a trematode species and different geographical populations of the same snail host species are known, including Fasciola\\n[26], [27]. Among lymnaeids, pronounced differences in susceptibility have been highlighted among snail populations encountered in close proximity [28], [29], and some snail populations have been mentioned to even show a total lack of susceptibility or resistance [29]\\u2013[31].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues1\", \"pone.0024567-Bargues2\", \"pone.0024567-Bargues4\"], \"section\": \"Introduction\", \"text\": \"However, the aforementioned different susceptibility phenomena in lymnaeids have to be considered with great caution. Many of these lymnaeid species involved in fascioliasis transmission maintain a confusing systematic-taxonomic status. When comparing different lymnaeid DNA sequences, several populations originally classified as belonging to different species showed identical DNA marker sequences, and other populations originally classified as pertaining to the same species presented different DNA marker sequences. Sometimes sequence differences were very few, suggesting intraspecific variability (different haplotypes). However, occasionally differences detected among populations classified as pertaining to the same species were numerous, sufficient as to consider different species involved. Moreover, the number of sequence differences between species sometimes appeared lower than that between populations of the same species [1], [14], [16]. This clearly underlines both the classification problems and the systematic-taxonomic confusion present in Lymnaeidae. Consequently, several susceptibility differences described could in fact be related to different lymnaeid species instead of different populations of the same lymnaeid species.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues1\", \"pone.0024567-MasComa5\", \"pone.0024567-Bargues4\", \"pone.0024567-Ueno1\", \"pone.0024567-Bargues5\", \"pone.0024567-MasComa6\", \"pone.0024567-MasComa1\", \"pone.0024567-Boray2\", \"pone.0024567-Pointier1\"], \"section\": \"Introduction\", \"text\": \"The crucial implications of lymnaeid vectors for fascioliasis transmission, epidemiology and control demonstrate the importance of developing new tools to facilitate specimen classification, genetic characterisation of natural populations and laboratory strains, and to elucidate the systematics and taxonomy of Lymnaeidae. The failure of all malacological and non-malacological tools applied to date suggests the analysis of DNA sequences and phylogenetic methods to be worthwhile. The first attempt made by a research collaboration of parasitologists, molecularists and malacologists was successful [1]. This success, together with the rapid realisation that locally restricted studies were insufficient because of the very large geographical distribution and spreading capacities of many lymnaeids belonging to very confusing lymnaeid groups, suggested early on that large, transboundary studies would be needed. In this sense, a worldwide lymnaeid molecular characterisation initiative was instigated [12], [16]. The great spreading capacity of lymnaeids means that sometimes not even the continental scale is sufficient, and intercontinental sequence comparisons are needed to classify specimens correctly. As an example: the sympatric Lymnaea viatrix and L. cubensis, which were noted to be involved in the human fascioliasis high hyperendemic area of the Bolivian Altiplano [32], later proved to only involve morphologically variable Galba truncatula of European origin [33]\\u2013[35]. The intercontinental spreading of lymnaeids and its role in fascioliasis dissemination is well known [2], [27], [36].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues3\", \"pone.0024567-Bargues4\", \"pone.0024567-Bargues5\", \"pone.0024567-Stothard1\", \"pone.0024567-Bargues1\", \"pone.0024567-Bargues2\", \"pone.0024567-Bargues3\", \"pone.0024567-MasComa6\", \"pone.0024567-Remigio1\", \"pone.0024567-Bargues7\", \"pone.0024567-Puslednik1\", \"pone.0024567-MasComa5\", \"pone.0024567-Bargues4\", \"pone.0024567-Gutierez2\"], \"section\": \"Introduction\", \"text\": \"Of the different DNA markers used hitherto in lymnaeids, the 18S gene of the nuclear ribosomal DNA (rDNA) appears to be too conserved and its few variable positions may only be useful at generic and suprageneric taxon levels [15], [16], [33], [37]. Ribosomal DNA ITS-2 and secondarily ITS-1 are the most useful sequences for studies at species level [1], [14], [15], [35], [38], [39], [40]. These two spacers are useful for: (i) classification of lymnaeid specimens, (ii) characterisation of lymnaeid intraspecific genetic interpopulational variablity to furnish the genetic base on which to understand fasciolid-lymnaeid specificity, different susceptibilities or compatibilities of geographical strains or even resistances, (iii) establishment of valid species and their geographical distributions, and (iv) assessment of species interrelationships to arrange a natural systematic-taxonomic classification, which will allow for an analysis of coevolution with fasciolids [12], [16]. Interestingly, one mutation at the level of the ITS-1 and another at ITS-2 have proved useful to distinguish between resistant and susceptible populations of P. columella in Cuba [41], although nothing evidently suggests that these mutations are linked to resistance/susceptibility.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues3\", \"pone.0024567-Puslednik1\", \"pone.0024567-Remigio2\", \"pone.0024567-Remigio4\", \"pone.0024567-Bargues8\", \"pone.0024567-MasComa7\", \"pone.0024567-MasComa5\"], \"section\": \"Introduction\", \"text\": \"Within mitochondrial DNA (mtDNA), only fragments of 16S and cox1 have been sequenced in lymnaeids [15], [40], [42]\\u2013[44]. Recent knowledge indicates that mtDNA markers, including both mitochondrial genes and the ribosomal 12S and 16S genes within the mitochondrial genome, should be used with great caution when dealing with lymnaeid species belonging to different genera and even those well-separated within the same genus [45]. Of particular concern is the saturation of nucleotide positions. Additionally, it has also been documented that incomplete gene sequences do not necessarily contain a sufficiently significant portion of the whole gene, i.e. parts of the gene presenting evolutionary hot spots may be missed [46]. Consequently, the use of mtDNA markers for this initiative is restricted to (i) sequence comparisons and phylogenetic analyses of only closely related species within the same genus, (ii) studies of intraspecific variability of species by sequence comparisons of individuals and populations, (iii) genetic characterisation of laboratory strains, (iv) studies on the spread of populations of a species, and (v) studies on genetic exchange between different neighboring populations [12].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-MasComa5\"], \"section\": \"Introduction\", \"text\": \"The present paper summarises results obtained in analyses of lymnaeid specimens of given populations originally ascribed to the main fascioliasis vector species G. truncatula in Europe, Asia, and Africa, and/or also to similar lymnaeid vector species belonging to the Galba/Fossaria group in North America, the Caribbean and South America. Such analyses were performed after obtaining unexpected results in experimental studies about lymnaeid susceptibility to Fasciola infection when assessing disease transmission characteristics in human hypo- to hyperendemic fascioliasis areas in the different continents. Multidisciplinary studies carried out to understand experimentally-tested, abnormal fasciolid susceptibility of these widely spread lymnaeid G. truncatula-like populations have taken more than 10 years. Characterisation studies have been made by nuclear rDNA and mtDNA sequences, phylogenetic tree reconstruction, phenotypic differentiation by shell morphometry, morphoanatomical characterisation, fecundation studies, ecological observations, and assessment of the geographical spread in correlation with historical events. These results demonstrate that another lymnaeid species, Lymnaea (s. l.) schirazensis, genetically distant but phenotypically very close, has always been confused with G. truncatula and/or other similar lymnaeid vector species in all these continents. The implications for fascioliasis are discussed, as this hitherto overlooked species has been distorting results of fasciolid specificity/susceptibility analyses as well as the geographical distribution of the disease. The existence of this G. truncatula-like lymnaeid species frequently present in animal fascioliasis endemic areas and usual in human fascioliasis endemic areas ought be henceforth considered to avoid misunderstandings concerning transmission. Moreover, results indicate that L. schirazensis can be used as a useful biomarker of foreign livestock introduction, a crucial aspect in fascioliasis spreading and emergence [12]. Additionally, L. schirazensis offers an outstanding new laboratory model for studies on genomics and proteomics about susceptibility/resistance in F. hepatica/lymnaeid interaction, an important field of applied research with disease control perspectives.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g001\"], \"section\": \"Lymnaeid snail material\", \"text\": \"The snail specimens studied (n\\u200a=\\u200a8572 specimens) were collected in the field along a ten-year period, from 20 lymnaeid populations present in geographical areas with human and/or animal fascioliasis endemicity of eight countries (Figure 1):\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567.s004\"], \"section\": \"Voucher specimens and taxonomic nomenclature aspects\", \"text\": \"The list of species (and subspecies) of molluscs and trematode parasites included in this study, according to the zoological nomenclature, is included in Table S1. The generic name Lymnaea is used here throughout in the broad taxonomic meaning (sensu lato\\u200a=\\u200as. l.).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues3\", \"pone.0024567-Bargues6\"], \"section\": \"DNA extraction\", \"text\": \"DNA was extracted from more than one specimen of a given population when this was deemed necessary for sequence verification. Only snails that appeared free of helminth infection were used in the molecular analyses. To reduce the risk of contamination of DNA from helminths further (which are more likely to be localized in other tissues), DNA was only isolated from the foot of each snail. Use of just the feet, rather than all the soft tissues, also prevented the development in the DNA pellets of the white flocculate substance (probably of polysaccharides) and melanic pigments that can inhibit Polymerase Chain Reaction (PCR) and cause amplification of non-specific products [15], [34].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues1\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues5\", \"pone.0024567-Sambrook1\"], \"section\": \"DNA extraction\", \"text\": \"Snail feet fixed in 70% ethanol were used for DNA extraction procedures. After dissection under a microscope, half of the foot was suspended in 400 \\u00b5l of lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulfate SDS) containing 500 \\u00b5g/ml Proteinase K (Promega, Madison, WI, USA) and digested for 2 hr at 55\\u00b0 C with alternate shaking each 15 min. Steps of the procedure were performed according to methods outlined previously [1], [15], [33]. Total DNA was isolated according to the phenol-chloroform extraction and ethanol precipitation method [47]. Each pellet was dried and resuspended in 30 \\u00b5l sterile TE buffer (pH 8.0). This suspension was stored at \\u221220\\u00b0 C until use.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues1\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues5\", \"pone.0024567-MasComa6\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues6\", \"pone.0024567-Bargues1\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues7\", \"pone.0024567-Simon1\", \"pone.0024567-Remigio2\", \"pone.0024567-Folmer1\"], \"section\": \"DNA sequence amplification\", \"text\": \"DNA sequences were amplified by PCR using 4\\u20136 \\u00b5l of genomic DNA for each 50 \\u00b5l PCR reaction, according to methods outlined previously [1], [15], [33], [35]. Each one of the five DNA markers were PCR amplified independently for each lymnaeid specimen and each PCR product was sequenced for a bona-fide haplotype characterization. A set of 8 conserved oligonucleotide primers was used for the amplification of five superimposed fragments of the 18S ribosomal RNA gene using specific primers and a standard protocol [15], [34] to amplify specific 18S rDNA regions. Ribosomal DNA spacers ITS-2 and ITS-1 were amplified using primers designed in conserved positions of 5.8S and 28S rRNA genes and 18S and 5.8S rRNA genes of several eukaryote Metazoa species, respectively [1], [15], [39]. The target 16S gene region was amplified by PCR using a set of universal primers [48]. Amplification procedures and thermal cycler conditions were carried out as previously described for lymnaeids [42]. A cox1 gene fragment was amplified using universal primers [49]. Amplifications were generated in a Mastercycle epgradient (Eppendorf, Hamburg, Germany), by 30 cycles of 30 sec at 94\\u00b0 C, 30 sec at 50\\u00b0 C and 1 min at 72\\u00b0 C, preceded by 30 sec at 94\\u00b0 C and followed by 7 min at 72\\u00b0 C for ITS-2 and ITS-1, and by 40 cycles of 30 sec at 90\\u00b0 C, 1 min at 48\\u00b0 C and 1 min at 72\\u00b0 C, preceded by 2.5 min at 94\\u00b0 C and followed by 10 min at 72\\u00b0 C for cox1. Ten \\u00b5l of each PCR product was checked by staining with ethidium bromide on 1% Nusieve\\u00ae GTG agarose (FMC) gel electrophoresis, using Molecular Weight Marker VI (Boehringer Mannheim) at 0.1 \\u00b5g DNA/\\u00b5l as control.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Sanger1\"], \"section\": \"DNA sequencing\", \"text\": \"The sequencing of the entire 18S rRNA gene, the complete rDNA ITS-2 and ITS-1, and the fragments of the mtDNA 16S and cox1 genes was performed on both strands by the dideoxy chain-termination method [50]. It was carried out with the Taq dye-terminator chemistry kit for ABI 373A and ABI 3700 capillary system (Perkin Elmer, Foster City, CA, USA), using PCR primers.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-MasComa5\", \"pone.0024567-Bargues4\", \"pone.0024567-Bargues7\"], \"section\": \"DNA haplotype nomenclature\", \"text\": \"The codes for the sequences obtained follow the standard nomenclature previously proposed for lymnaeid snails [12], [16], [39]. Note that haplotype codes are only definitive in the case of complete sequences. When dealing with fragments or incomplete sequences, haplotype codes are provisional.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Thompson1\", \"pone.0024567-Tamura1\", \"pone.0024567-Staden1\", \"pone.0024567-Swofford1\"], \"section\": \"Sequence alignments\", \"text\": \"Sequences were aligned using CLUSTAL-W version 1.8 [51] and MEGA 4.0 [52], and assembly was made employing Staden Package [53]. Subsequently, minor corrections were manually introduced for a better fit of nucleotide correspondences in microsatellite sequence regions. Homologies were performed using the BLASTN programme from the National Center for Biotechnology information website (http://www.ncbi.nlm.nih.gov/BLAST). Genetic distances were measured using parameters provided by PAUP v.4.0 b10 [54].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567.s004\"], \"section\": \"Sequence comparisons\", \"text\": \"The following sequences from GenBank-EMBL were used for comparative analyses (in the following, names of taxa according to articles in which sequences were described; see Table S1 for systematic-taxonomic notes):\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues5\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues6\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues8\", \"pone.0024567-Bargues9\", \"pone.0024567-Bargues5\"], \"section\": \"\", \"text\": \"18S rRNA gene: complete sequences of Lymnaea (Lymnaea) stagnalis (GenBank Accession Number Z73984), Lymnaea (Stagnicola) palustris (Z73983), Omphiscola glabra (Z73982), and Galba truncatula (Z73985) [33]; L. cubensis (Z83831) [15], [34], L. viatrix and L. neotropica (both species with the same sequence AM412222) [15], and L. humilis (FN182190) [45], all four from respective type localities; L. cousini and L. meridensis (both species with the same sequence FN598151), and Pseudosuccinea columella (FN598152) [55]; Radix auricularia (Z73980) and R. balthica (Z73981) [33]. Other incomplete sequences available at GenBank were not used to avoid problems in comparative sequence analyses.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues1\", \"pone.0024567-Bargues2\", \"pone.0024567-Remigio1\", \"pone.0024567-Bargues1\", \"pone.0024567-Bargues3\", \"pone.0024567-MasComa6\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues8\", \"pone.0024567-Bargues9\", \"pone.0024567-Bargues9\"], \"section\": \"\", \"text\": \"rDNA ITS-2: L. (S.) palustris palustris (AJ319620), L. (S.) palustris turricula (AJ319618), L. (S.) fuscus (AJ319621), and Catascopia occulta (AJ319642) [1], [14]; C. catascopium (AF013143), C. emarginata (AF013141, AF013142), C. elodes (AF013138), and Hinkleyia caperata (AF013139) [38]; G. truncatula H1 (AJ296271), H2 (AJ243017), and H3 (\\u200a=\\u200aL. viatrix sensu Ueno et al., 1975;\\u200a=\\u200aL. cubensis sensu Ueno et al., 1975) (AJ272051) [1], [15], [35]; L. cubensis (AM412223), L. viatrix (AM412224), L. neotropica (AM412225) [15], L. humilis (FN182191) [45], L. cousini (FN598153), and L. meridensis (FN598154) [55], all six sequences from respective type localities; P. columella (FN598156) [55].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues7\", \"pone.0024567-Remigio1\", \"pone.0024567-Bargues1\", \"pone.0024567-Bargues3\", \"pone.0024567-MasComa6\", \"pone.0024567-Bargues7\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues8\", \"pone.0024567-Bargues9\", \"pone.0024567-Bargues9\"], \"section\": \"\", \"text\": \"rDNA ITS-1: L. (S.) palustris palustris (AJ626849), L. (S.) palustris turricula (AJ626853), L. (S.) fuscus (AJ626856), and C. occulta (AJ626858) [39]; C. catascopium (AF013143), C. emarginata (AF013142), C. elodes (AF013138), and Hinkleyia caperata (AF013139) [38]; G. truncatula HA (AJ243018), HB (AJ296270), and HC (\\u200a=\\u200aL. viatrix sensu Ueno et al., 1975;\\u200a=\\u200aL. cubensis sensu Ueno et al., 1975) (AJ272052) [1], [15], [35], [39]; L. cubensis (AM412226), L. viatrix (AM412227), L. neotropica (AM412228) [15], L. humilis (FN182193) [45], L. cousini (FN598157), and L. meridensis (FN598159) [55], all six sequences from respective type localities; P. columella (FN598160) [55].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Remigio3\", \"pone.0024567-Wethington1\", \"pone.0024567-Bargues8\"], \"section\": \"\", \"text\": \"mtDNA 16S rRNA gene: Fossaria bulimoides (AF485657), F. obrussa (AF485658), S. elodes (AF485652), and Stagnicola bonnevillensis (AF485655) [43]; F. bulimoides (EU038315) and S. elodes isolate 44106 (EU038305) [56]; L. cubensis (FN182204) and L. humilis (FN182195) [45].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Wethington1\", \"pone.0024567-Remigio4\", \"pone.0024567-Bargues3\", \"pone.0024567-Albrecht1\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues8\", \"pone.0024567-Bargues3\", \"pone.0024567-MeraySierra1\", \"pone.0024567-Bargues8\", \"pone.0024567-Bargues9\", \"pone.0024567-Remigio4\", \"pone.0024567-Bargues9\"], \"section\": \"\", \"text\": \"mtDNA cox1 gene: S. elodes (EU038352) [56]; S. elodes (AY227368), F. bulimoides (AY227367), and Austropeplea tomentosa (AY227365) [44]; G. truncatula from Spain (AM494011) [15] and Germany (EU818799) [57]; L. cubensis from Cuba (AM494009\\u2013type locality) [15] and the USA (FN182205) [45], L. viatrix (AM494010\\u2013type locality), L. neotropica from Peru (AM494008\\u2013type locality) [15] and Argentina [58], L. humilis (FN182197-9\\u2013type locality) [45], L. cousini (FN598161\\u2013type locality, and FN598162-3) and L. meridensis (FN598164\\u2013type locality) [55]; P. columella from Australia (AY227366) [44] and Puerto Rico (FN598165) [55].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Winnepennickx1\", \"pone.0024567-DeRijk1\"], \"section\": \"Representation of the 18S rRNA secondary structure\", \"text\": \"The previously published secondary structure prediction for Limicolaria kambeul 18S rRNA [59], based on the general eukaryote 18S rRNA secondary structure [60], was used and extended to encompass lymnaeid sequences.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Swofford1\", \"pone.0024567-Posada2\"], \"section\": \"Phylogenetic inference\", \"text\": \"Phylogenies were inferred from DNA sequences using maximum-likelihood (ML) estimates with PAUP [54]. ML parameters such as model, base frequencies, transition/transversion ratio (ts/tv), the shape parameter for the gamma distribution, and the proportion of invariant sites, were optimized using the hierarchical likelihood ratio test (hLRT) and the Akaike information criterion (AIC) [61,62), implemented in Modeltest 3.7 [63]. Starting branch lengths were obtained using the least-squares method with ML distances.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Saitou1\", \"pone.0024567-Ronquist1\"], \"section\": \"Phylogenetic inference\", \"text\": \"To provide an assessment of the reliability of the nodes in the ML tree, three methods were used. First, a bootstrap analysis using 1000 replicates was made with heuristic search in PAUP. Second, a distance-based phylogeny using the neighbour-joining (NJ) algorithm [64] with ML pairwise distances was obtained. Statistical support for the nodes was evaluated with 1,000 bootstrap replicates, with and without removal of gapped positions. Third, a Bayesian phylogeny reconstruction procedure was applied to obtain posterior probabilities (BPP) for the nodes in the ML tree, by using the same evolutionary model as above, implemented in MrBayes 3.1 [65] with four chains for 1,000,000 generations and trees were sampled every 100 generations. The first 1,000 trees sampled were ruled out (\\u201cburn-in\\u201d), and clade posterior probabilities (PP) were computed from the remaining trees. Alternative methods of phylogenetic reconstruction allowing for an evaluation of the support for each node were also applied. A distance-based phylogeny using the NJ algorithm with LogDet distances was obtained. Statistical support for the nodes was evaluated with 1,000 bootstrap replicates.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-MasComa7\", \"pone.0024567-Lin1\", \"pone.0024567-Ballard1\", \"pone.0024567-Bargues4\"], \"section\": \"Phylogenetic inference\", \"text\": \"Due to different limitations recently shown by mtDNA markers for interspecific sequence analyses in invertebrates [46], [66], [67], phylogenetic reconstruction by combined sequences data sets was made from ribosomal and mitochondrial markers separately. Combined sets analysed were: a) ITS-1 and ITS-2, considered as the markers to fit best for the analysis of relationships between species, as has already been verified in Lymnaeidae [16]; b) 18S, ITS-1 and ITS-2, to increase the support for evolutionarily older divergence nodes; c) 16S and cox1, to evaluate mtDNA genome information; and d) cox1 gene, by using both the three and only the first two codon positions to assess potential saturation.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-DeJong1\", \"pone.0024567-DeJong1\"], \"section\": \"Phylogenetic inference\", \"text\": \"Phylogenetic analyses were performed using reference sequences of lymnaeid DNA (see species and Acc. Nos. in list noted above in chapter of sequence comparisons), after adding the following sequences stored at databases: ITS-2: Radix auricularia H1 (AJ319628) and R. balthica H1 (AJ319633); ITS-1: R. auricularia HA (JF922878) and R. balthica HA (JF922879); 16S: L. (L.) stagnalis (AF485659), L. (S.) palustris (U82082), G. truncatula (HQ283236), L. viatrix (HQ283239), L. humilis HA and HB (FN182195, FN182196), L. cousini (HQ283237), L. meridensis HA, P. columella (U82073) and R. auricularia (AF485659). cox1: L. (L.) stagnalis (EU818795), L. (S.) palustris (EU818801), R. auricularia (EU818800). The intergenic region sequence (AY030361) [68] including both ITSs of a planorbid species, Biomphalaria pfeifferi, was used as outgroup. For the combined sets using 18S and 16S, B. alexandrina 18S (BAU65225), ITSs (AY030371) and 16S (AY030204) [68] were used for outgroups, similarly as B. alexandrina cox1 (AF199110).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Samadi1\", \"pone.0024567-Valero3\"], \"section\": \"Phenotypic study\", \"text\": \"Shells and egg clusters of lymnaeids were measured, according to traditional malacological methods [19], using a computerised image-analysis system (CIAS) [69]. This system is based on a DXC-930P colour video camera (Sony, Tokyo) fitted to a stereomicroscope, and connected to computer running image analysis software (ImageProH Plus 4.5; Media Cybernetics Inc., Silver Spring, MD).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Anonymous1\"], \"section\": \"Phenotypic study\", \"text\": \"Egg clusters were obtained from living lymnaeids experimentally maintained in climatic chambers. Living egg clusters were measured with CIAS. For egg clusters, cluster roundness (CR\\u200a=\\u200aCP2/4\\u03c0CA) measurements were used to quantify the cluster shape. It is a measure of how circular an object is (the expected perimeter of a circular object divided by the actual perimeter). A circular object will have a roundness of 1.0, while more irregular objects will have larger values [70].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-MasComa6\"], \"section\": \"Liver fluke experimental infection assays\", \"text\": \"Isolates of F. hepatica from Poland and Peru, and F. gigantica from Egypt and Vietnam, maintained in the laboratory according to previously described methods [35], were used for experimental infection assays of the laboratory-reared lymnaeids.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t001\", \"pone-0024567-g001\"], \"section\": \"DNA sequences\", \"text\": \"Sequence characteristics are noted in Table 1. Data includes length and GC/AT content, obtained for each one of the five DNA markers analysed in the populations of L. schirazensis from the twenty localities of eight countries (Figure 1) with their corresponding haplotype codes and GenBank Accession Nos.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t001\", \"pone-0024567-g002\"], \"section\": \"18S rRNA gene\", \"text\": \"All of the 20 populations of L. schirazensis presented the same 18S sequence (Table 1). When comparing this 18S sequence with that of G. trucatula, a total of 19 variable positions appeared. They included 6 ts, 2 tv and 11 insertions/deletions (indels) in a 1853 bp-long pairwise alingment, most differences being located in the variable Helix E-10 of the V2 region according to the secondary structure (Figure 2).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues5\", \"pone.0024567-Bargues5\", \"pone.0024567-Bargues6\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues8\", \"pone.0024567-Bargues9\", \"pone.0024567-Bargues3\", \"pone.0024567-Bargues9\"], \"section\": \"Nucleotide differences in a total of 64 variable positions found in the complete 18S rDNA sequence of the lymnaeid species compared and their location in the secondary structure.\", \"text\": \"Helix, Position and Difference number\\u200a=\\u200anumbers to be read in vertical. Position\\u200a=\\u200anumbers refer to positions obtained in the alignment made with MEGA 4.0. Identical\\u200a=\\u200a.; Indel\\u200a=\\u200a\\u2212. Shaded area corresponds to Helix E10-1 of the variable area V2 where differences in the 18S rRNA gene of Lymnaeidae are concentrated [33]. GenBank Accession Nos.\\u200a=\\u200aZ73980\\u2212Z73985 [33]; Z83831 [34]; AM4122222 [15]; FN182190 [45]; FN598151- FN598152 [55]; L. schirazensis from present paper. Sequence correspondences: * L. cubensis, L. viatrix, L. humilis and L. cousini without definitive genus ascription; 18S identical in L. viatrix and L. neotropica\\n[15]; 18S identical in L. cousini and L. meridensis\\n[55].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g002\"], \"section\": \"18S rRNA gene\", \"text\": \"A multiple 18S sequence alignment was 1867 bp long. It included: several Galba/Fossaria vector species such as L. cubensis, L. viatrix (the sequence being identical to that of L. neotropica), G. truncatula, L. humilis, and L. cousini (sequence identical to that of L. meridensis); the peculiar species P. columella; three representatives of stagnicolines (L. (L.) stagnalis, L. (S.) palustris and O. glabra); and two of the Radix group (R. auricularia and R. balthica). This alignment showed a total of 64 variable nucleotide positions (3.43% nucleotide divergence). Thirty-two of these 64 polymorphic sites appeared grouped in the short sequence between positions 206 and 266, which corresponds to the helix E10-1 of the variable area V2 of the secondary structure (Figure 2).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t001\"], \"section\": \"Second internal transcribed spacer rDNA ITS-2\", \"text\": \"Two different ITS-2 haplotypes were found among the L. schirazensis populations analysed (Table 1). Their pairwise comparison showed only 8 polymorphic sites, corresponding to 8 indels caused by the tetranucleotide microsatellite repeat TGCT, present twice in haplotype 1 between positions 128 and 135 of the alignment but absent in haplotype 2.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t002\"], \"section\": \"Second internal transcribed spacer rDNA ITS-2\", \"text\": \"The very high number of nucleotide differences detected in the pairwise comparisons of these two ITS-2 sequences with the three ITS-2 haplotypes of G. truncatula available at GenBank is noteworthy. In the different alignments performed, with a bp length varying between 469 to 477, a total of 130\\u2013139 differences appeared, including 18\\u201319 ts, 11 tv and 101\\u2013109 indels (Table 2).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567.s005\"], \"section\": \"Second internal transcribed spacer rDNA ITS-2\", \"text\": \"When comparing the L. schirazensis ITS-2 sequences with the other species of the Galba/Fossaria group, the pairwise ITS-2 distance matrix obtained with PAUP (only parsimony informative sites considered) showed that the numbers of nucleotide differences were unexpectedly high in all cases. The lowest of these numbers appeared when comparing with G. truncatula, although rather high (64\\u201366). Regarding L. humilis, L. cousini and L. meridensis, it ranged from 99 to 118. It appeared to be similarly high (121\\u2013136) with regard to L. cubensis, L. viatrix and L. neotropica. Nucleotide differences were also numerous when L. schirazensis was compared with stagnicolines and Pseudosuccinea (Table S2).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t001\"], \"section\": \"First internal transcribed spacer rDNA ITS-1\", \"text\": \"Two different ITS-1 haplotypes were also found among the L. schirazensis populations analysed (Table 1). The pairwise comparison of these two sequences showed only 3 polymorphic sites, corresponding to two insertions in positions 296 and 297 (AA or --) and to one mutation in position 443 (A or T) in haplotypes A and B, respectively.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t002\"], \"section\": \"First internal transcribed spacer rDNA ITS-1\", \"text\": \"A very high number of nucleotide differences also appeared in the pairwise comparisons with the three ITS-1 haplotypes of G. truncatula. In the different alignments performed, with a bp length varying from 558 to 560, a total of 134\\u2013138 differences appeared, including 31\\u201332 ts, 21\\u201323 tv and 81\\u201383 indels (Table 2).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567.s006\"], \"section\": \"First internal transcribed spacer rDNA ITS-1\", \"text\": \"When comparing L. schirazensis ITS-1 sequences with the other species of the Galba/Fossaria group, the pairwise ITS-1 distance matrix obtained with PAUP showed that the numbers of nucleotide differences were also very large. The comparison with G. truncatula furnished the second lowest number (81\\u201384), after that of the comparison with L. meridensis (77\\u201380). Regarding L. humilis and L. cousini, it ranged from 87 to 92, and it appeared to be similarly high (83\\u201392) with regard to L. cubensis, L. viatrix and L. neotropica. The number of nucleotide differences increased considerably when L. schirazensis was compared with stagnicolines (132\\u2013143) and Pseudosuccinea (129\\u2013130) (Table S3).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t001\"], \"section\": \"16S rRNA gene of the mtDNA\", \"text\": \"Two different sequences of the 16S fragment were found (Table 1). They differred by only 2 mutations (C/A in position 85 and A/G in position 305 of their pairwise alignment, in HA/HB, respectively).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g003\"], \"section\": \"16S rRNA gene of the mtDNA\", \"text\": \"Nucleotide comparison with other Galba/Fossaria species showed a total of 46 variable positions (10.75%), of which 31 were mutations (7.24%) and 15 were indels (3.50%). This alignment demonstrated that L. schirazensis was different from any other Galba/Fossaria at the level of this mtDNA gene (Figure 3). In pairwise comparisons, minimum differences were 18 mutations (4.2%) when compared with L. humilis, and a maximum of 22 mutations (5.14%) appeared in comparisn with L. cubensis. Nucleotide differences were more numerous when comparing with the stagnicolines S. elodes and S. bonnevillensis. The four species alignment showed a total of 56 variable positions (12.93%), 47 of which were mutations (10.85%) and 9 indels (2.08%) (alignment not shown).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Bargues8\", \"pone.0024567-Remigio3\", \"pone.0024567-Wethington1\", \"pone.0024567-Bargues8\"], \"section\": \"Variable positions showed by the mtDNA 16S sequence fragment in a 433-bp-long alignment including the two haplotypes of Lymnaea schirazensis and other Galba/Fossaria species.\", \"text\": \"Numbers (to be read in vertical) refer to positions obtained in the alignment made with MEGA 4.0. Identical\\u200a=\\u200a. ; Indel\\u200a=\\u200a\\u2212 . Haplotype codes only provisional due to incomplete sequences of the gene. L. cubensis 16S-HB (FN182204), L. humilis 16S-HA (FN182195) and L. humilis 16S-HB (FN182196) [45]; F. bulimoides (AF485657) and F. obrussa (AF485658) [43]; * the sequence ascribed to the stagnicoline C. elodes (EU038305) [56] concerns in fact a Galba/Fossaria species-see analysis in [45].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t001\", \"pone-0024567-t003\", \"pone.0024567.s007\"], \"section\": \"mtDNA cytochrome c oxidase subunit I cox1\", \"text\": \"Four different cox1 sequences were obtained (Table 1). When aligned, a total of 661 positions were conserved and only 11 were variable, comprising 2 parsimony informative and 9 singleton sites (Table 3). When comparing with species of the Galba/Fossaria group and other proximal lymnaeid species available at GenBank, whose cox1 fragment sequences were similar in length to those obtained in the present study, the higher number of nucleotide differences appeared evident in a pairwise cox1 distance matrix (Table S4). A comparison with more distant species such as stagnicolines (S. elodes) and Radix group (Austropeplea tomentosa) was not necessary.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g004\"], \"section\": \"mtDNA cytochrome c oxidase subunit I cox1\", \"text\": \"The amino-acid sequence of that COX1 gene fragment was 224 aa long. A pairwise comparison showed a 100% identity between the three L. schirazensis haplotypes cox1-a, cox1-b and cox1-c, and only one amino-acid difference (isoleucine/threonine, respectively) in position 96 with L. schirazensis haplotype cox1-d from Ba\\u00f1os del Inca, Peru. The provisional haplotypes COX1-I and COX1-II were assigned, respectively. The comparison with the amino acid sequence of other species of the Galba/Fossaria group and other proximal lymnaeid species showed that COX1-I of L. schirazensis was identical to that of other species, such as L. viatrix, L. humilis and L. meridensis. However, L. schirazensis COX1-II appeared to be unique (Figure 4).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g005\"], \"section\": \"Combined ITS-1 and ITS-2 sets\", \"text\": \"This single data-set generated a robust tree, indicating phylogenetic accordance between the two spacers. The ML model best fitting this data-set was HKY85+I, using a ts/tv ratio of 2 (kappa\\u200a=\\u200a3.8979086), base frequencies for A, C, G and T of 0.21534, 0.26862, 0.22661 and 0.28943, respectively, and a proportion of invariable sites\\u200a=\\u200a0.087. To assess the reliability of the nodes in the ML tree (Figure 5 A), a bootstrap analysis using 1,000 replicates was made using branch-swapping algorithm (tree-bisection-reconnection TBR) with heuristic search and the neighbour-joining (NJ) algorithm with the ML pairwise distances in PAUP. Finally, a Bayesian phylogeny reconstruction procedure was applied to obtain posterior probabilities (BPP) for the nodes in the ML tree with MrBayes 3.1.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567.s004\"], \"section\": \"Phylogenetic trees of lymnaeid species studied based on maximum-likelihood (ML) estimates: A) data set of ITS-1 and ITS-2, with the planorbid B. pfeifferi as outgroup (\\u2212Ln\\u200a=\\u200a10016.27013); B) same data set of ITS-1 and ITS-2 with B. pfeifferi as outgroup, after adding Radix species (\\u2212Ln\\u200a=\\u200a10078.46520).\", \"text\": \"Scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; b: bootstrap with ML reconstruction using PAUP with 1000 heuristic replicates; c: Bayesian posterior probability with ML model using MrBayes. See Table S1 for systematic-taxonomic notes.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g005\"], \"section\": \"Combined ITS-1 and ITS-2 sets\", \"text\": \"In the ML tree obtained (Figure 5 A), P. columella appeared basal to two large groupings, namely stagnicolines (including both Palaearctic and Nearctic species), and the Galba/Fossaria clade which comprised the F. hepatica main vector species. Lymnaea schirazensis appeared included within the latter. However, contrary to what would be expected, L. schirazensis did not cluster together with G. truncatula, of Old World origin, or other morphologically similar lymnaeids such as the New World L. cubensis, L. viatrix and L. neotropica. Surprisingly, it appeared linked to the Nearctic L. humilis and the two Neotropical L. cousini and L. meridensis, within a relatively well supported branch (82/78/88 in NJ/ML/BBP).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g005\"], \"section\": \"Combined ITS-1 and ITS-2 sets\", \"text\": \"The topology obtained with the NJ algorithm using LogDet distances (figure not shown) was identical to that shown by the ML tree (Figure 5 A).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g005\"], \"section\": \"Combined ITS-1 and ITS-2 sets\", \"text\": \"When adding two Radix species (data matrix of 24 taxa with 1,767 characters), the ML model best fitting was HKY85+G+I, using a ts/tv ratio of 1.45 (kappa\\u200a=\\u200a2.872483), base frequencies for A, C, G and T of 0.22764, 0.25354, 0.23176 and 0.28705, respectively, a shape parameter (alpha)\\u200a=\\u200a0.99, and a proportion of invariable sites\\u200a=\\u200a0.13. In the ML tree obtained (Figure 5 B), Radix species clustered independently in a branch basal to all the remaining lymnaeids. Unexpectedly, the location of L. schirazensis changed, now becoming basal to the other Galba/Fossaria members which clustered together although with very low supports. The topology of the NJ LogDet tree was similar (figure not shown), but with L. schirazensis in a subclade with G. truncatula showing low bootstrap (only 59).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g006\"], \"section\": \"Combined 18S, ITS-1 and ITS-2 set\", \"text\": \"This three ribosomal marker data set (matrix of 17 taxa and 3,540 characters) also generated a robust tree. The ML model best fitting was HKY85+I, using a ts/tv ratio of 1.48 (kappa\\u200a=\\u200a2.9601982), base frequencies for A, C, G and T of 0.22150, 0.25100, 0.25820 and 0.26930, respectively, and a proportion of invariable sites\\u200a=\\u200a0.44. In the ML tree obtained (Figure 6 A), L. schirazensis appeared clustering together with G. truncatula (79/81/- in NJ/ML/BPP), and the branch including L. humilis, L. cousini and L. meridensis appeared as the sister group inside the Galba/Fossaria clade. The topology obtained with the NJ algorithm using LogDet distances (figure not shown) was identical.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567.s004\"], \"section\": \"Phylogenetic trees of lymnaeid species studied based on maximum-likelihood (ML) estimates: A) data set of 18S, ITS-1 and ITS-2, with B. alexandrina as outgroup (\\u2212Ln\\u200a=\\u200a13171.38533); B) data set of 16S and cox1, with B. alexandrina as outgroup (\\u2212Ln\\u200a=\\u200a5282.96177).\", \"text\": \"Scale bar indicates the number of substitutions per sequence position. Support for nodes a/b/c: a: bootstrap with NJ reconstruction using PAUP with ML distance and 1000 replicates; b: bootstrap with ML reconstruction using PAUP with 1000 heuristic replicates; c: Bayesian posterior probability with ML model using MrBayes. See Table S1 for systematic-taxonomic notes.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g006\"], \"section\": \"Combined 16S and cox1 set\", \"text\": \"The data matrix analysed contained 19 taxa and 1,118 characters. The ML model best fitting was HKY85+G+I, using a ts/tv ratio of 1.78 (kappa\\u200a=\\u200a5.083228), base frequencies for A, C, G and T of 0.33500, 0.10560, 0.12210 and 0.43730, respectively, a shape parameter (alpha)\\u200a=\\u200a0.39, and a proportion of invariable sites\\u200a=\\u200a0.35. The ML tree (Figure 6 B) showed L. schirazensis haplotypes clustering together with the branch of L. humilis, L. cousini and L. meridensis, although with low support values (60/\\u2013/60). The topology furnished by the NJ algorithm using LogDet distances (figure not shown) was similar, with that clade supported by a 64% bootstrap.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567-Kster1\"], \"section\": \"Type locality\", \"text\": \"Shiraz, Iran [71].\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g007\", \"pone-0024567-t004\", \"pone.0024567.s008\"], \"section\": \"Shell\", \"text\": \"The shell was brownish to reddish light brown, thin-walled, elongated conical, usually with four regular convex whorls and up to 5.5 whorls in the longer specimens (Figure 7). The whorls were somewhat inflated, slightly shouldered, with silky and longitudinally striated surface and separated by a deep, well-marked suture, increasing rather slowly in diameter. The columella was straight, unfolded, and the umbilicus open. The last or body whorl was almost \\u00be times as high as the shell height, presenting a slight twisting trend along its basal part visible in the biggest shells when viewed dorsally, and which was due to the enlargement of the basal lip of the peristome (in the way of Pseudosuccinea columella). The spire was pointed. The aperture was elongatedly oval, slightly oblique, mid-sized and wider at the base. The thin peristome was patent throughout, the umbilicus partially covered by a widened columellar lip. The shell showed a tendency to be approximately one and a half to two times as long as it is wide, and its aperture tends to be slightly less than half as long as the shell. Measurements and calculated ratios of natural and experimental populations were noted (see Table 4 and Table S5, respectively).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g008\", \"pone-0024567-g008\", \"pone-0024567-g008\", \"pone-0024567-g008\"], \"section\": \"External morphological characteristics\", \"text\": \"The cephalopedeal mass was pale greyish. The eyes were black, relatively big in size (Figure 8 A\\u2013C). Tentacles were elongate, slender, pyramidal, with narrow base (Figure 8 A\\u2013E). The mantle roof was dark, from dark brown to blackish throughout, with small unpigmented white-greyish round spots including several tiny circles at the beginning of the border of the pulmonary region and a few scattered further away in between the initial large round spots (Figure 8 F\\u2013I). The border of the mantle was light grey. The black pigmentation of the hypopeplear region of the mantle roof gave a dark appearance to the shell of living specimens by transparency (Figure 8 D,E). This dark appearance did not depend on the characteristics of the natural habitat, as it was maintained across the different laboratory-reared snail generations (quite the opposite of what happens with several darkish populations of other lymnaeid species under experimental conditions).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g009\", \"pone.0024567.s001\", \"pone-0024567-g009\"], \"section\": \"Anatomy\", \"text\": \"Morphoanatomical features were studied (see Figure 9 and Figure S1). The renal tube extended straight from the pericardial region toward the mantle collar, diagonally across the roof of the palial cavity and parallel to the pulmonary vein and renal vein. It was a straight tube, lined with a folded epithelium, white to white-yellowish, that tapered, smoothed and became transparent distalward until the level of the septum between pulmonary and hypopeplear cavities. In that place, it turned with an almost right-angle to form a short ureter (which lacked the two distinct flexures present in other close species) and finally opened behind the pneumostome. A very slender muscle thread appeared lengthwise on the ventral surface of the renal tube. A layer of very thin transverse parallel muscle fibers gave the inner side of the pulmonary wall a densely striated appearance (Figure 9 A,B).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g009\"], \"section\": \"Anatomy\", \"text\": \"The ovotestis showed a lobulate surface formed by several acini, with a collecting canal which continued into the ovispermiduct. The latter presented a short and very thin proximal segment emptying into the seminal vesicle, an expanded portion of bosselated surface, which narrowed down into a slender distal segment ending in the carrefour surrounded by the albumen gland, oviduct and spermiduct (Figure 9 C\\u2013F).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g009\", \"pone.0024567.s001\", \"pone-0024567-g009\", \"pone.0024567.s001\"], \"section\": \"Anatomy\", \"text\": \"The voluminous albumen gland covered the carrefour and the origin of a bosselated, transverse tubular oviduct which followed a somewhat convolute course, described a nearly complete circle in contact with the albumen gland, and continued into a striated, oblong nidamental gland (Figure 9 C,D and Figure S1 A,B). The initial part of the nidamental gland increased in width to keep the diameter throughout its length or even slightly enlarged in its distal part, to subsequently narrow to give rise to a wrinkled-walled uterus followed by a short vagina. The latter shows a first conical narrowing part and a final short tubular part (without any bulbous swelling or sphincter-like thickening present in other close species) (Figure 9 E\\u2013G and Figure S1 A,B), opening into the female genital pore with a slightly thickened lip. All parts of the female reproductive system proximal to the vagina itself showed a pronounced width.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone.0024567.s001\", \"pone-0024567-g009\", \"pone.0024567.s001\"], \"section\": \"Anatomy\", \"text\": \"The yellowish spermatheca (Figure S1 A,B) had an oval body and gave rise to a thin, relatively long spermathecal duct which emerged laterally from the spermatheca instead of from its terminal extremity. The almost uniformly thin, smooth-walled spermathecal duct extended diagonally between the nidamental gland and the prostate until joining the final part of the uterus and the beginning of the vagina. The spermathecal duct was not dilated at its distal end (Figure 9 E\\u2013G and Figure S1 B).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g009\", \"pone.0024567.s001\", \"pone-0024567-g009\", \"pone.0024567.s001\", \"pone-0024567-g009\", \"pone.0024567.s001\"], \"section\": \"Anatomy\", \"text\": \"Emerging from the carrefour, the spermiduct showed an initial slightly dilated part with a granular outer surface slendering into a uniformly thin, straight duct which ran on the ventral surface of the nidamental gland. The distal portion of the spermiduct widened to form a prostate of granular surface, relatively small size, narrower than the nidamental gland. The small, oblong, light grey prostate gland varied in shape from inverted-pear-like to elongated-potato-like, without ventral lengthwise fissure (Figure 9 E,F and Figure S1 C). In cross section, the prostate showed a simple, slit-like lumen (lacking internal folds known in other lymnaeids) (Figure 9 H and Figure S1 D). The prostate gave distally rise to a smooth-walled vas deferens which ran shortly in contact with the vagina and extended until looping caudalward. After this loop, the vas deferens became a uniformly thin, long duct to finally merge into a curved, penial sheath or phallotheca (Figure 9 E,F and Figure S1 E,F).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g009\", \"pone.0024567.s001\"], \"section\": \"Anatomy\", \"text\": \"The penis sheath was regularly cylindrical, with a very slightly thicker proximal part (which showed no patent circlet of minute knobs or ring of papillae described in close species). The penis sheath was about a little bit more than half as long than the praeputium. The maximum width of the praeputium was around two or even three times thicker than the penis sheath. The shape of the praeputium was usually elongate-conical, gradually narrowing to terminate in the male genital pore, although it also appeared cylindrical thoughout most of its length and only tapering distally (Figure 9 E,F and Figure S1 E,F). This conical shape of the praeputium appeared to be an exception among the typical conical praeputium shape in Lymnaeidae. The length of the praeputium measured 0.73\\u20131.96 mm (mean 1.13 mm) while that of the penis sheath was 0.36\\u20130.94 mm (mean 0.72 mm), with a praeputium length/penis sheath length ratio of 1.20\\u20132.23 (mean 1.60).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g010\", \"pone-0024567-t005\"], \"section\": \"Egg clusters\", \"text\": \"Shape variability of egg clusters and eggs was studied (Figure 10), and measurements and calculated ratios were noted (Table 5). Egg clusters were saccular, transparent, with a very thin outer membranous wall. The shape of these egg clusters showed an evident trend from kidney- to banana-like; the more curved and relatively more elongated and narrow the more numerous were the eggs inside. Small clusters containing only very few eggs (2\\u20135) appeared rounded to oval, but the elongate-curved shape became soon apparent when the egg number increased. Eggs were from spherical to slightly ovoid, with a uniformly thick outer shell, usually appearing well separated from each other.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g011\", \"pone-0024567-g011\", \"pone-0024567-g011\", \"pone-0024567-g011\", \"pone-0024567-g011\", \"pone-0024567-g011\", \"pone-0024567-g011\", \"pone-0024567-g011\", \"pone-0024567-g011\"], \"section\": \"Habitat\", \"text\": \"In nature, this species was almost never found in water, nor even close to water edges, sometimes up to 1 m or farther from it (Figure 11 I). Outside water, it was usually found on the soil surface under grass besides slow running large rivers (Figure 11 B) or even under relative high bushes completely away from sunhine (Figure 11 H). The smallest amount of water to keep humidity was sufficient for this species to maintain populations, as in ditches of rural paths and dirt tracks (Figure 11 L,N) or even artificial cement canals in gardens (Figure 11 A) and drainage canalizations around buildings and dwellings (Figure 11 K). Man-made small irrigation canals around plant cultures also appeared to offer appropriate conditions for its development (Figure 11 C,D), as well as large drainage cement canals with almost no vegetation (Figure 11 E). Worth mentioning was its presence on mud and livestock footprints in and around animal farms (Figure 11 M).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g011\"], \"section\": \"Habitat\", \"text\": \"Besides this link to antropophilic habitats and livestock-inhabited places, it was also found, however, in more silvatic habitats. Examples were specimens under the dense vegetation of the edges of fast running waters in mountain rivers and streams, where populations undoubtedly derived from dragging and floods by the water from upper areas (Figure 11 F,J,G).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-g011\"], \"section\": \"Habitat\", \"text\": \"Worth mentioning was that specimens of L. schirazensis sometimes appeared so mixed or close to one another with specimens of G. truncatula, that one was convinced to deal with a population of only one species, e.g. in Spain (Figure 11 D,E).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t006\"], \"section\": \"Selfing and egg cluster laying\", \"text\": \"Egg cluster laying was verified to occur in all of the specimens from different populations maintained individually isolated after hatching (Table 6). Age and size when laying began differed somewhat from one individual to another within a population, less in populations of the New World (Mexico) than in those of the Old World (Egypt, Spain). Despite range overlap, those from Mexico proved to be markedly sexually precocious in age when compared to those from Egypt and Spain. Snail size when laying began did not show a similar correlation, suggesting that development speed and reaching a sufficient size may be more important for sexual activity.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t006\"], \"section\": \"Selfing and egg cluster laying\", \"text\": \"Comparison of the measurements of the first and first five clusters laid by each individual snail also showed that slight differences may be found according to the populations. Results indicated that there is no gradual increase in cluster size nor in the number of eggs per cluster within the laying of a snail specimen. Thus, sometimes the first egg cluster laid by a snail individual was larger and contained more eggs than those laid afterwards (2nd to fifth clusters) by the same specimen (Table 6). However, somewhat larger clusters including a slightly larger number of eggs/cluster were laid by older and larger snail specimens (up to 20 and 22 eggs/cluster in specimens from Egypt and Spain, respectively).\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t006\", \"pone-0024567-t007\", \"pone-0024567-g012\"], \"section\": \"Selfing and egg cluster laying\", \"text\": \"Snail life span was relatively short, of around 2.5\\u20136.8 months. It includes a sexually active period of cluster laying lasting a total of 39\\u2013161 days, sometimes even only very few days before death. However, the final postlaying period may reach slightly more than 1.5 months in given specimens (Table 6 and Table 7; Figure 12). Both life span and laying period appeared to be longer in Old World (Egypt, Spain) specimens than in the New World (Mexico) specimens.\"}, {\"pmc\": \"PMC3183092\", \"pmid\": \"21980347\", \"reference_ids\": [\"pone-0024567-t006\", \"pone-0024567-t007\"], \"section\": \"Comparison showing interpopulational differences of mean characteristics of life span and selfing reproduction capacity of isolatelly-maintained Lymnaea schirazensis specimens of four populations from different geographical origins, experimentally followed up from the day of their individual isolation immediately after hatching.\", \"text\": \"Populations studied: Albufera of Valencia, Valencia, Spain; Tiba, Delengate district, Egypt; Escuela A. Obrego, La Trinidad Tepango, Atlixco, Puebla, Mexico; and Jiutepec, Morelos, Huauchinango, Mexico. n\\u200a=\\u200a10 snail speci

Metadata

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