Pattern of Genetic Divergence of Mitochondrial DNA Sequences in Biomphalaria tenagophila Complex Species Based on Barcode and Morphological Analysis

Neotropical region has a high diversity of Biomphalaria (Pulmonata: Basommatophora: Planorbidae) fauna with at least twenty-six of the world‘s estimated thirtyseven species recorded (Carvalho,2008). Nearly one –third of these species occur in freshwater ecosystems related to the main drainage river basins in Brazil (Estrada et al.,2006; Teodoro,2010),where Biomphalaria glabrata (Say,1818),Biomphalaria tenagophila (Orbigny,1835) and Biomphalaria straminea (Dunker,1848) have serious impact on human health for these species host Schistosoma mansoni Sambon 1907 (Figure 1).


Pattern of Genetic Divergence of Mitochondrial DNA Sequences in
Biomphalaria tenagophila Complex Species Based on Barcode and Morphological Analysis 295 The morphological characters of the genital organs employed by the malacologist WL Paraense permits all of us to correctly identify the taxonomic units of the tenagophila complex.But,in fact,identifying Biomphalaria species solely on morphological characters leads to a less precise identification of these taxonomical groups,which are found to be difficult to diagnose in a malacological work routine.
The following examples may illustrate how morphological or technical variations can lead to erroneous species identification with serious epidemiological consequences.Example 1: Within the species of planorbid described in Brazil,a B. glabrata is the only species to feature pigmented renal ridge (Paraense and Deslandes 1959).This character is known to be the only to set the difference between B. glabrata (the most important host species of S. mansoni in the Americas) and B. tenagophila,responsible for the transmission of schistosomiasis in the southeastern region of Brazil.Pepe et al. (2009) reports that a pigmentation-like may appear associated to the renal ridge in B. t. guaibensis,as a result from an intraspecies variation.Such diagnosis would trigger an unnecessary alert once B.t.guaibensis is not an intermediary host of S. mansoni.Variation in the pattern of the pigmentation of the renal ridge may also reflect the influences of the freshwater substrate types leading us to an erroneous identification of all Biomphalaria species.Here we exemplified the two major aggravating factors in a morphological identification system: morph variation within the species and variation caused by interaction of the organism with the environment.Example 2: According to Paraense (1984) an essential condition for a clear distinction of B.tenagophila from the subspecies B. t. guaibensis "is that the animal body is preserved for dissection in a well relaxed condition",that is,for the identification of the subspecies,more rigorous laboratorial methods of looseness need to be used instead of the methods utilized in the current identification of mollusks.If the B.t. guaibensis subspecies were dispersed,it would be difficult to have a morphological differential diagnosis of these subspecies.The intrinsic difficulties to the system of morphological identification of organisms limit the description of taxonomic discreet units,although they lead to the development of auxiliary molecular techniques to overcome these difficulties (Estrada et al.,2006;Carvalho,2008).Species of planorbid snails belonging to the genus Biomphalaria have received increasing attention in molecular systematic and phylogeographic studies in recent years (Vidigal et al. 2000(Vidigal et al. ,2004;;Carvalho et al. 2001;DeJong et al. 2001DeJong et al. ,2003;;Mavárez et al. 2002a,b;Morgan et al. 2002;Pointier et al. 2005;Tuan and Santos,2007).The development of genetic markers and the databases generated in such studies can be further used for multiple purposes,of both theoretical and applied interest: species identification,analysis of population genetic structure and speciation patterns,inference of common/independent origins of susceptibility to Schistosoma,detection of natural hybridization,tracking of geographical expansions and biological invasions,evaluation of genetic differentiation between phenotypic variants and/or geographic isolates,amongst others.Genetic markers used in Biomphalaria studies include mitochondrial DNA (Spatz et al. 1999;DeJong et al. 2001DeJong et al. ,2003;;Mavárez et al. 2002b;Pointier et al. 2005).The latter is especially useful for species identification.The dispersal and colonizing ability of B. tenagophila highlights the need to monitor the spread of this species within and outside the neotropical region.The identification of B. tenagophila in Kinshasa,Africa (Pointier,2005) and Romania (Majoros et al. 2008) certainly caused great concern to public health authorities.
For Biomphlalaria species two important characteristics play an important role to successfully establish snail populations in a environment outside the native range of the species: self-fertilization and desiccation.B.tenagophila species are simultaneous hermaphrodites snails with the possibility of both self and cross-fertilization (Paraense,1955;Tuan & Simões,1989;Guimarães,2003).Such a complex mode of reproduction might probably increase colonization since new populations can arise from just one snail by self-fertilization.On the other hand reproduction alone is not enough to establish a species because snails still must be able to survive conditions outside their native range.One important aspect to B. tenagophila survival it is the ability to withstand desiccation in response to water shortage (Tuan & Simões,1989;Ohlweiler & Kawano,2002) that greatly increases the possibility of recolonization after a population crash or colonization of new environments.These must be a major source of genetic diversity amongst the same species creating patchily distributed populations that can be easily distinguished by molecular markers.In this study,we obtained mitochondrial COI and 16SrRNA sequences from B. tenagophila,B.t. guaibensis,and B. occidentalis specimens collected in the São Paulo state,and Rio Grande do Sul state,Brazil to estimate the divergence of each species amongst the three members of the tenagophila complex and evaluate the potential of COI and 16S rRNA mitochondrial genes for identification of closely related species.

Molecular analysis
B. tenagophila and B. occidentallis snails were collected along the banks of different streams of Paranapanema,Ribeira do Iguape,Paraíba do Sul,Tietê,and Litoral Basins at São Paulo,Brazil.B.t. guaibensis specimens were collected at Rio Grande do Sul,Brazil (Table 1).Two samples from Argentina were also analyzed.A total of 66 specimens were used in this study.Prior to molecular analysis snails were identified through morphological analysis,according to Deslandes (1959) and Paraense (1975Paraense ( ,1981Paraense ( ,and 1984)).Total genomic DNA was extracted from foot tissue of individual snails using DNeasy Tissue Kits (Qiagen®) and was preserved as DNA vouchers specimens in the DNA voucher collection of the Laboratory of Molecular Biology and Biochemical at SUCEN.Amplification of COI gene was attempted using LCO-1490 and HCO-2198 (Folmer et al.,1994).Amplifications (in 50µl of total volume) consisted of 10-100 ng of DNA,0.2 mM of each dNTP,0.10µM of each primer and 1 U of Taq DNA polymerase (BioLabs) with the supplied buffer.The following PCR temperature profile was used: a initial 3 min step at 95ºC for denaturation,25 iterations of 1 min at 95ºC,1 min at 47ºC and 1 min 30 sec at 72ºC,and a final extension step for 7 min at 72ºC.The amplification profile of 16SrRNA fragment included an initial denaturation step at 94°C for 2 min.and 35 cycles of 30 sec at 94°C,30 sec at 54°C and 1 minute at 72°C,and a final step at 72°C for 5 minutes.Amplification of 16S gene was attempted using a Palumbi forward and reverse primer (Palumbi, 1996).PCR products were checked by agarose gel electrophoresis,cleaned using QIAquick PCR Purification Kits (Qiagen®).Cycle-sequencing of PCR products were carried out using terminal primers given above and the BigDye Terminator v3.1 kit of Applied Biosystems (ABI),purified using DyeEx-Kit (Qiagen®) according to the modified protocol and sequenced on an ABI 310 automated sequencer.
Table 1.Collecting data,code and geographical identification of the specimen used in molecular study.

Morphology analysis
The morphological analysis was based on 10 dissected specimens B. tenagophila from Arambaré,Rio Grande do Sul (lot DPE 9011); 13 dissected specimens B. t. guaibensis from Porto Alegre,Rio Grande do Sul (lot DPE 9008,Figure 3) and 31 dissected specimens B. occidentalis from Tremembé,São Paulo (lot DPE 9028).Only large and adult specimens were used for dissection and anatomic study.

Molecular analysis
The evolutionary relationships of tenagophila complex were inferred using Neighbor-Joining method for COI and 16SrRNA mitochondrial genes (Figure 4).The evolutionary distances depicted as branch lengths were computed using the Kimura 2p method using 550 nucleotides positions for the COI dataset and 314 nucleotide positions for the 16S rRNA dataset.The tenagophila complex sequences were retrieved in two well distinct and bootstrap supported branches: one with B. occidentalis clustering with B. t. guaibensis,and another distinct branch with the nominal B.tenagophila sequences.For each component that belongs to tenagophila complex we obtained intraspecific identical sequences that support morphological taxonomy.Two distinct groups of B. tenagophila were recovered in each analysis (COI and rRNA16S).The Figure 5 shows that no overlap exists between intraspecific and interspecific distribution distance calculated under Kimura 2P.Indeed,the distributions of the COI and 16S genetic distances are very similar (Figure 5).In order to examine the ability of the barcoding methodology to identify the three Biomphalaria taxa we plotted the intra and interspecific divergence of COI and 16S using a threshold of 3% minimum interspecific genetic variation and 3% maximum intraspecific genetic variation (Figure 6).According the 3% threshold value for within and between species variation four quadrants are defined.Species on quadrant I (dark grey) are well-defined species.Groups on quadrant II (white colored) are composite species.Species on quadrant III (medium grey) are recent divergent species,and species on quadrant IV (light grey) are misidentified species.B. tenagophila,B.t. guaibensis and B. occidentalis had no significant differences among the number of whorls.The species had typically six to eight whorls.Otherwise,the shell aperture would be shorter and narrower in B.t guaibensis than in the other two species analyzed (Figure 7).Biomphalaria snails can be distinguished based on differences in the shape and size of their male and female internal organs as described in Figure 8.The differences between the species were mainly found in the male and female systems.The anatomical characters that clearly groups B.tenagophila and B. t. guaibensis in a same taxon is a well developed vaginal pouch located in the vaginal wall of this species,and also the shape and size of the prepuce and the volume of prostata (Table 5,Figure 9).In all the three species the lateral were characterized with three cuspids.Small subcusps appear on lateral teeth near the margins given a serrated appearance for the radulae.

Discussion
Phylogenetic analysis using short sequences of the mitochondrial COI and 16SrRNA genes resulted in trees with high support values morphologycally pairs of species B. tenagophila and the subspecies B. t. guaibensis,and the B. occidentalis and B. t guaibensis,assigned as tenagophila species complex (Figure 4).The molecular evidence thus strongly suggests that B. occidentalis and B.t. guaibensis are part of a clade of closely related species with a minimal genetical divergence between them (Table 2).
The result obtained here is in perfect agreement with our observations with respect to the resemblance between guaibensis and occidentalis morphology,both thin and elongated species when compared to tenagophila species.For taxonomically purposes,the vaginal pouch places B. tenagophila and B.t. guaibensis closer to each other (Paraense,1984).The vaginal pouch represents a character easy to score unambiguously (1 for presence 0 for absence) but instead of inferring a close relationship between B.tenagophila and B.t guaibensis,the vaginal pouch could also be considered a case of homoplasy and might not have great confidence and definition for such a complex taxonomic groups as in other gastropod groups (Collin,2003).To sustain such hypothesis,further additional sequences from other sites are clearly required.
Our sequence data (COI,16S) revealed significant intraspecific variation for B. tenagophila.Two major branches were resolved by phylogenetic analysis,one of which -G1-with very low internal variation (pi=0.0000for COI and 16S) and a second branch -G2-with a significant degree of internal genetic differentiation amongst sequences (pi=0.00587for COI and pi= 0.00995 for 16S).
The extremely low levels of genetic variation in G1 branch exists amongst populations across a broad geographic range area,including Ourinhos,Pindamonhangaba and Taubate.All these sites presented were in the past active areas of transmission of schistosomiasis (Piza,1972;Silva,1985),subjected to intensive use of moluscicide measure that resulted in cycles of extinction followed by recolonization of B. tenagophila (Tuan,1996).Whereas G1 population occurs in historical unstable sites,G2 population inhabits freshwater sites at São Paulo (Registro,Juquia) with fragments of the Brazilian Atlantic Rain Forest,one of the most important hot-spots of genetic diversity in the world (Carnaval et al. 2009).The significant differentiation between G1 and G2 B. tenagophila populations (Fst COI= 0.82; Fst 16S=0.77)and the retention of even a small genetic diversity in G2 populations could be due to historical signatures since Atlantic coastal regions played an important role as a climatic refugee during the last Pleistocene (Pfenninger and Posada,2009).A taxonomist working with a series of snails of this species collected over a broad geographical range certainly would not recognize any significant morphological variation.This shows the lack of resolution of morphology and that DNA barcoding data can direct us to what we need to look further into: biodiversity.According to Moritz and Cicero (2004) "accurate diagnosis" by a short COI DNA sequence,"depends on low intraspecific variation compared with that between species" So we can conclude that COI and 16S may be used for detection of species in B. tenagophila complex species (Figure 5).Furthermore,mtDNA lineages found in B. tenagophila may help explain heterogeneous patterns of schistosomiasis transmission.

Fig. 3 .
Fig. 3.A natural freshwater habitat at Porto Alegre where B. tenagophila guaibensis were collected.29° 59' 52 3''S, 51° 15' 38.6' W. Snails were identified according to Paraense (1975).Soft parts were preserved under fixation as vouchers.The identification of the mollusks was done under the stereomicroscopy (Leica MZ 95) and was photographed with the image Program IMS 50.We took into consideration the morphology of the shell and soft parts,especially the reproductive system.Three radulae and three jaws of the B. occidentalis,B.tenagophila and B. t. guaibensis were extracted and examined under a Scanning Electron Microscope LEO 440 at the Museum of the Zoology,São Paulo University (MZUSP).
Fig. 4. Optimal trees inferred for a 550 fragment COI mtDNA(A),sum of branch length= 0.09775397,and for a 314 nucleotides 16SrRNA,sum of branch length= 0.07712277.Bootstrap values (1000 replicates) are shown next to the branches.

Fig. 5 .
Fig. 5. Distribution of intraspecific variation genetic distance (in blue bars),and interspecific distance (in green bars) estimated by Kimura´s 2 -parameter model (K2P) for all the species studied

Fig. 7 .
Fig. 7. Shell.A: B. t. guaibensis Biomphalaria tenagophila, C: B. occidentalis Fig. 8. Reproductive System from Biomphalaria tenagophila.GA: albumen gland,GN: nidamental gland,OV: ovotestis,OVI: oviduct,OVIS: ovispermiduct,PP: prepuce,PR: prostate,PS: penial sheath,SP: spermatheca,SPE: spermiduct,UT: uterus,VA: vagina,VD: vas deferens,VP: vaginal pouch, VS: seminal vesicle.The hermaphrodite part did not present differences between B.tenagophila,B.t.guaibensis and B. occidentalis.The differences between the species were mainly found in the male and female systems.The anatomical characters that clearly groups B.tenagophila and B. t. guaibensis in a same taxon is a well developed vaginal pouch located in the vaginal wall of this species,and also the shape and size of the prepuce and the volume of prostata (Table5,Figure9).

Fig. 9 .
Fig. 9. Detail of reproductive system.A,D and G: B. tenagophila,B,E and H: B. t. guaibensis,C,F and I: B. occidentalis.A,B and C: Penis; D,E and F: Vaginal complex; G,H and I: Prostate The radulae of B tenagophila,B. t. guaibensis and B. occidentalis consisted of a fused jaw (Figure 10) to which the teeth are attached.

Table 5 .
Differences among B. tenagophila species complex based on reproductive parameters