Complete phylogeny of Micrathena spiders suggests multiple dispersal events among Neotropical rainforests, islands and landmasses, and indicates that Andean orogeny promotes speciation.

The Neotropical region is the most diverse on the planet, largely owing to its mosaic of tropical rainforests. Multiple tectonic and climatic processes have been hypothesized to contribute to generating this diversity, including Andean orogeny, the closure of the Isthmus of Panama, the GAARlandia land bridge and historical connections among currently isolated forests. Micrathena spiders are diverse and widespread in the region, and thus a complete phylogeny of this genus allows the testing of hypotheses at multiple scales. We estimated a complete, dated phylogeny using morphological data for 117 Micrathena species and molecular data of up to five genes for a subset of 79 species. Employing eventc-based approaches and biogeographic stochastic mapping while considering phylogenetic uncertainty, we estimated ancestral distributions, the timing and direction of dispersal events and diversification rates among areas. The phylogeny is generally robust, with uncertainty in the position of some of the species lacking sequences. Micrathena started diversifying around 25 Ma. Andean cloud forests show the highest in-situ speciation, while the Amazon is the major dispersal source for adjacent areas. The Dry Diagonal generated few species and is a sink of diversity. Species exchange between Central and South America involved approximately 23 dispersal events and started ~20 Ma, which is consistent with a Miocene age for the Isthmus of Panama closure. We inferred four dispersal events from Central America to the Antilles in the last 20 Myr, indicating the spiders did not reach the islands through the GAARlandia land bridge. We identified important species exchange routes among the Amazon, Andean cloud forests and Atlantic forests during the Plio-Pleistocene. Sampling all species of the genus was fundamental to the conclusions above, especially in identifying the Andean forests as the area that generated the majority of species. This highlights the importance of complete taxonomic sampling in biogeographic studies.

Evaluating the boundaries of marine biogeographic regions of the Southwestern Atlantic using halacarid mites (Halacaridae), meiobenthic organisms with a low dispersal potential.

AIM: We evaluated traditional biogeographic boundaries of coastal marine regions in Southwestern Atlantic using DNA sequence data from common, rocky-shore inhabiting, marine mites of the genera Agauopsis and Rhombognathus, family Halacaridae. METHODS: We investigated geographic population genetic structure using CO1 gene sequences, estimated divergence times using a multigene dataset and absolute time-calibrated molecular clock analyses, and performed environmental niche modeling (ENM) of common marine mite species. RESULTS: Agauopsis legionium has a shallow history (2.01 Ma) with four geographically differentiated groups. Two of them corresponded to the traditional Amazonian and Northeastern ecoregions, but the boundary between the two other groups was inferred at the Abrolhos Plateau, not Cabo Frio. Rhombognathus levigatoides s. lat. was represented by two cryptic species that diverged 7.22 (multilocus data) or 10.01 Ma (CO1-only analyses), with their boundary, again at the Abrolhos Plateau. ENM showed that A. legionium has suitable habitats scattered along the coast, while the two R. levigatoides cryptic species differ considerably in their niches, especially in parameters related to upwelling. This indicates that genetic isolation associated with the Abrolhos Plateau occurred in both lineages, but for the R. levigatoides species complex, ecological niche specialization was also an important factor. MAIN CONCLUSIONS: Our study suggests that the major biogeographic boundary in the Southwestern Atlantic lies not at Cabo Frio but at the Abrolhos Plateau. There two biogeographically relevant factors meet (a) changes in current directions (which limit dispersal) and (b) abrupt changes in environmental parameters associated with the South Atlantic Central Waters (SACW) upwelling (offering distinct ecological niches). We suggest that our result represents a general biogeographic pattern because a barrier at the Abrolhos Plateau was found previously for the fish genus Macrodon (phylogeographic data), prosobranch mollusks, ascidians, and reef fishes (community-level data).

Molecular phylogeny of marine mites (Acariformes: Halacaridae), the oldest radiation of extant secondarily marine animals.

The family Halacaridae comprises more than one thousand mostly marine or rarely freshwater species. Many are predacious, but among marine mites, some genera evolved the ability to feed on macroalgae. We inferred a time-calibrated phylogeny based on 18S rDNA, 28S rDNA, and Cytochrome oxidase I (5,143 nt aligned) and all non-monotypic halacarid subfamilies plus a representative outgroup set (72 taxa). The family Halacaridae was rendered as the sister-group of Parasitengona, diverging 321.5, 264.0-381.3 Ma and radiating 271.3, 221.7-324.2 Ma (median, HPD). Thus, marine mites represent the oldest known extant animal lineage that secondarily invaded the sea, with the marine turtles being the second oldest such lineage (crown group 212.3, 194.9-231.4 Ma). Two freshwater mite lineages, represented by Limnohalacarus (219.2, 165.9-274.6) and Porohalacarus (175.3, 118.5-233.1), were inferred mutually non-monophyletic, suggesting two independent invasions to freshwater. The conventional subfamily Rhombognathinae (macroalgae feeders) was not recovered as monophyletic, with Metarhombognathus-Rhombognathides, restricted to the Northern Hemisphere, originating 177.5, 134.8-223.3 Ma and diversifying 88.3, 32.7-152.3 Ma. This is congruent to a previous hypothesis of their northern origin prior to the opening of the Norwegian Sea (58 Ma). Our phylogeny indicates the need for reclassification of the traditional subfamilies and suggests that previous molecular results (e.g., Rhombognathus deeply nested in Copidognathinae) is an analytical artifact due to a chimeric sequence.

Predicting the distribution of Omalonyx (Mollusca: Pulmonata: Succineidae) species from literature review, museum databases and new sampling efforts in Brazil / Predição da distribuição de Omalonyx (Mollusca: Pulmonata: Succineidae) a partir da revisão da literatura, dados de museu e novo esforço amostral no Brasil

Abstract Accurate distributional information is crucial for studies on systematics, biodiversity and conservation. To improve the knowledge regarding the geographical distribution of Omalonyx in South America, we present updated information based on data from a literature review, institutional collections and malacological surveys. All this information composed the dataset used to predict species distribution employing the Maximum Entropy Algorithm (MaxEnt). The model was run using data on species distribution, altitude and bioclimatic variables (WorldClim database). The model had consistent performance, and areas presenting similar conditions to areas where the species were recorded were considered areas of occurrence. The predicted occurrence areas included those that were already surveyed and those that are considered potential occurrence areas. The results demonstrate that the genus has widespread distribution in the Neotropical region and occurs in the tropical, temperate and arid regions of South America and Lesser Antilles. Omalonyx spp. were recorded in all South American countries and hydrographic regions. However, in some countries, there were only isolated records (ex: Colombia and Ecuador). Here, we also present the first record of Omalonyx spp. in four Brazilian States (Acre, Rondônia, Piaui, and Amapá). The genus was found in all hydrographic regions within Brazil and among 27 federative unities; it was absent from only two unities (Roraima State and Distrito Federal). This work contributes to the knowledge on Omalonyx spp. distribution and provides an important basis for the work of ecologists and taxonomists.

Resumo A informação precisa sobre a distribuição é crucial para os estudos sobre sistemática, biodiversidade e conservação. Para melhorar o conhecimento sobre a distribuição geográfica de Omalonyx na América do Sul, apresentamos informações atualizadas com base em dados de revisão de literatura, coleções institucionais e pesquisas malacológicas. Toda essa informação compôs o conjunto de dados usado para predição da distribuição de espécies empregando o Algoritmo de Entropia Máxima (MaxEnt). O modelo foi executado usando dados de distribuição de espécies, altitude e variáveis bioclimáticas (banco de dados WorldClim). O modelo apresentou um desempenho consistente e as áreas que apresentaram condições semelhantes às áreas onde as espécies foram registradas, foram consideradas áreas de ocorrência. As áreas de ocorrências previstas incluíram aquelas que já foram pesquisadas e aquelas que são consideradas áreas de ocorrência potencial. Os resultados demonstram que o gênero tem uma distribuição Neotropical ampla e que ocorre nas regiões tropical, temperada e árida da América do Sul e nas Pequenas Antilhas. Omalonyx spp. foram registradas em todos os países e bacias sul-americanas. No entanto, em alguns países, apenas registros isolados foram encontrados (ex: Colômbia e Equador). Aqui, também apresentamos o primeiro registro de Omalonyx spp. em quatro estados brasileiros (Acre, Rondônia, Piauí e Amapá). O gênero foi encontrado em todas as regiões hidrográficas no Brasil e nas 27 unidades federativas; sendo ausente em apenas duas unidades federativas (Estado de Roraima e Distrito Federal). Esse trabalho contribui para o conhecimento da distribuição das espécies de Omalonyx e fornece uma importante base para trabalhos de ecólogos e taxonomistas.

First record of Corbicula largillierti (Philippi 1844) in the Paraíba River Basin and potential implications from water diversion of the São Francisco River / Primeiro registro de Corbicula largillierti (Philippi 1844) na bacia do Rio Paraíba e implicações potenciais com a transposição das águas do Rio São Francisco

Corbicula largillierti is a native mollusk from China. In Brazil, this species was first recorded in the Pantanal of Mato Grosso. This short communication reports the occurrence of C. largillierti for the first time in the Paraíba river basin (Brazilian semi-arid), and also considers the risk of introduction of other molluscs invaders in this basin due to the diversion of water from the São Francisco River. Densities of individuals ranged from 33 to 65 ind.m-2 (maximum values of 484 ind.m-2) in coarse sediment (gravel, 2-4 mm). The diversion of waters from the São Francisco river can lead to the introduction of new species, enhancing ecological problems in the Paraiba river basin.

Corbicula largillierti é um molusco nativo da China. No Brasil esta espécie foi registrada primeiramente no Pantanal do Mato Grosso. Esta nota registra a primeira ocorrência de C. largillierti na bacia do Rio Paraíba (semiárido brasileiro). Considera também os riscos potenciais de introdução de outros moluscos invasores nesta bacia devido è transposição das águas do Rio São Francisco. As densidades do molusco variaram de 33 a 65 ind.m-2 (atingindo valor máximo de 484 ind.m-2) em sedimentos grossos (cascalho, 2-4 mm). A transposição das águas do Rio São Francisco pode ocasionar a introdução de novas espécies exóticas potencializando problemas ecológicos na bacia do Rio Paraíba.

Strong spatial structure, Pliocene diversification and cryptic diversity in the Neotropical dry forest spider Sicarius cariri.

The Brazilian Caatinga is part of the seasonally dry tropical forests, a vegetation type disjunctly distributed throughout the Neotropics. It has been suggested that during Pleistocene glacial periods, these dry forests had a continuous distribution, so that these climatic shifts may have acted as important driving forces of the Caatinga biota diversification. To address how these events affected the distribution of a dry forest species, we chose Sicarius cariri, a spider endemic to the Caatinga, as a model. We studied the phylogeography of one mitochondrial and one nuclear gene and reconstructed the paleodistribution of the species using modelling algorithms. We found two allopatric and deeply divergent clades within S. cariri, suggesting that this species as currently recognized might consist of more than one independently evolving lineage. Sicarius cariri populations are highly structured, with low haplotype sharing among localities, high fixation index and isolation by distance. Models of paleodistribution, Bayesian reconstructions and coalescent simulations suggest that this species experienced a reduction in its population size during glacial periods, rather than the expansion expected by previous hypotheses on the paleodistribution of dry forest taxa. In addition to that, major splits of intraspecific lineages of S. cariri took place in the Pliocene. Taken together, these results indicate S. cariri has a complex diversification history dating back to the Tertiary, suggesting the history of dry forest taxa may be significantly older than previously thought.

Short-term toxicity of ammonia, sodium hydroxide and a commercial biocide to golden mussel Limnoperna fortunei (Dunker, 1857).

Macrofouling bivalves are considered an ecological and technological problem worldwide. Control measures have been researched with Limnoperna fortunei, but without success. The aim of the manuscript is to test some alternatives to regulate this harmful invasive mollusk. Mortality and behavioral response (shell gaping) of Limnoperna fortunei exposed to three chemical compounds were evaluated. Values for LC50 96h were: 0.25 (0.24-0.27)mg/L NH3-N, 11.10 (7.45-16.55) mg/L MXD-100 and 88.51 (74.61-105.01)mg/L NaOH. Reduced gaping was observed beginning at concentrations of 0.31mg/L (NH3-N), 100mg/L (MXD-100) and 160mg/L (NaOH) and increased above these values. The percentage of individuals gaping after two hours at LC50 96h differed significantly (χ(2)=79.9; DF=3; p<0.001) in MXD-100 (50%), NaOH (0%), NH3-N (96.7%) and the controls (93.3%). This study contributes to the understanding of the relationship between toxicity and behavioral effects of some toxicants in L. fortunei.

Size polymorphism in alleles of the myoglobin gene from biomphalaria mollusks.

Introns are common among all eukaryotes, while only a limited number of introns are found in prokaryotes. Globin and globin-like proteins are widely distributed in nature, being found even in prokaryotes and a wide range of patterns of intron-exon have been reported in several eukaryotic globin genes. Globin genes in invertebrates show considerable variation in the positions of introns; globins can be found without introns, with only one intron or with three introns in different positions. In this work we analyzed the introns in the myoglobin gene from Biomphalaria glabrata, B. straminea and B. tenagophila. In the Biomphalaria genus, the myoglobin gene has three introns; these were amplified by PCR and analyzed by PCR-RFLP. Results showed that the size (number or nucleotides) and the nucleotide sequence of the coding gene of the myoglobin are variable in the three species. We observed the presence of size polymorphisms in intron 2 and 3; this characterizes a homozygous/heterozygous profile and it indicates the existence of two alleles which are different in size in each species of Biomphalaria. This polymorphism could be explored for specific identification of Biomphalaria individuals.

Angiostrongylus costaricensis (Nematoda: Protostrongylidae): migration route in experimental infection of Omalonyx sp. (Gastropoda: Succineidae).

Angiostrongylus costaricensis can infect several mollusks, and its migration route in intermediate hosts has been studied only in Sarasinula marginata. To verify the susceptibility of Omalonyx sp. as an intermediate host of A. costaricensis and to analyze the nematode migration route, individuals were infected with stage 1 larvae. Obtained stage 3 larvae were orally inoculated in mice, and after 30 days, adult worms and stage 1 larvae were recovered, demonstrating Omalonyx susceptibility and suitability to infection. To define the parasite migration routes, specimens of Omalonyx with 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 2 days, 5 days, 10 days, 12 days, 15 days, 20 days, 21 days, 25 days, 28 days, and 30 days of infection were fixed and serially sectioned. Histological sections were stained with hematoxylin-eosin. The results were compared to those described in S. marginata. Oral and cutaneous infections were noted. After the penetration, larvae were retained, mainly in the fibromuscular tissue, by hemocytes, or they spread to the whole organism through the circulation, following the anatomical structure of the vasculature. The perilarval hemocyte reaction in Omalonyx was more intense until stage 2 larva instar, decreasing in the presence of stage 3 larvae. Differences in some aspects of hemocyte reaction between S. marginata and Omalonyx exemplify interspecific peculiarities in snail response to the same parasite.

Multiplex PCR for both identification of Brazilian Biomphalaria species (Gastropoda: Planorbidae) and diagnosis of infection by Schistosoma mansoni (Trematoda: Schistosomatidae).

A simple and single-step technique based on multiplex PCR (multiplex polymerase chain reaction) has been developed for simultaneous identification of Brazilian Biomphalaria species, the intermediate hosts of Schistosoma mansoni, and their diagnosis of infection by the trematode. We used species-specific primers directed both to the internal transcribed spacer 2 (ITS2) of ribosomal DNA from 3 of the S. mansoni host species and to the mitochondrial DNA (mtDNA) from the trematode. Those primers were used simultaneously in a single multiplex-PCR reaction, and template DNA was obtained from S. mansoni-infected and noninfected snails. The results were visualized in silver stained polyacrylamide gels, revealing the presence of specific bands. The methodology has shown to be efficient, fast, and reproducible for Biomphalaria species identification and diagnosis of snails infected by S. mansoni during prepatent periods.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae).

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni.

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina.

Polymerase chain reaction and restriction fragment length polymorphism of cytocrome oxidase subunit I used for differentiation of Brazilian Biomphalaria species intermediate host of Schistosoma mansoni

The intermediate hosts of Schistosoma mansoni, in Brazil, Biomphalaria glabrata, B. tenagophila and B. straminea, were identified by restriction fragment length polymorphism analysis of the mitochondrial gene cytochrome oxidase I (COI). We performed digestions with two enzymes (AluI and RsaI), previously selected, based on sequences available in Genbank. The profiles obtained with RsaI showed to be the most informative once they were polymorphic patterns, corroborating with much morphological data. In addition, we performed COI digestion of B. straminea snails from Uruguay and Argentina

Genetic variability in Brazilian populations of Biomphalaria straminea complex detected by simple sequence repeat anchored polymerase chain reaction amplification

Biomphalaria glabrata, B. tenagophila and B. straminea are intermediate hosts of Schistosoma mansoni, in Brazil. The latter is of epidemiological importance in the northwest of Brazil and, due to morphological similarities, has been grouped with B. intermedia and B. kuhniana in a complex named B. straminea. In the current work, we have standardized the simple sequence repeat anchored polymerase chain reaction (SSR-PCR) technique, using the primers (CA)8RY and K7, to study the genetic variability of these species. The similarity level was calculated using the Dice coefficient and genetic distance using the Nei and Li coefficient. The trees were obtained by the UPGMA and neighbor-joining methods. We have observed that the most related individuals belong to the same species and locality and that individuals from different localities, but of the same species, present clear heterogeneity. The trees generated using both methods showed similar topologies. The SSR-PCR technique was shown to be very efficient in intrapopulational and intraspecific studies of the B. straminea complex snails

Characterization of Biomphalaria orbignyi, Biomphalaria peregrina and Biomphalaria oligoza by polymerase chain reaction and restriction enzyme digestion of the internal transcribed spacer region of the RNA ribosomal gene

The correct identification of Biomphalaria oligoza, B. orbignyi and B. peregrina species is difficult due to the morphological similarities among them. B. peregrina is widely distributed in South America and is considered a potential intermediate host of Schistosoma mansoni. We have reported the use of the polymerase chain reaction and restriction fragment length polymorphism analysis of the internal transcribed spacer region of the ribosomal DNA for the molecular identification of these snails. The snails were obtained from different localities of Argentina, Brazil and Uruguay. The restriction patterns obtained with MvaI enzyme presented the best profile to identify the three species. The profiles obtained with all enzymes were used to estimate genetic similarities among B. oligoza, B. peregrina and B. orbignyi. This is also the first report of B. orbignyi in Uruguay

Further studies on the molecular systematics of Biomphalaria snails from Brazil

The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecular identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails.

Identificação específica e filogenia de moluscos neotropicais do gênero Biomphalaria através de técnicas de biologia molecular / Specific identification and phylogeny of snails of the Neotropical genus Biomphalaria using techniques of molecular biology

A identificação morfológica dos moluscos do gênero Biomphalaria é dificultada, principalmente, pela semelhança e extensa variação intraespecífica observada nos caracteres morfológicos utilizados na identificação clássica desses moluscos. Com o objetivo de contornar esses problemas, introduzimos metodologias moleculares como ferramentas adicionais à identificação morfológica. A análise de polimorfismo de seqüência da região espaçadora interna (ITS1 + 5.8S + ITS2) do gene codificador do RNAr de Biomphalaria, amplificada através da PCR (reação em cadeia da polimerase) e posterior digestão com enzimas de restrição (RFLP-polimorfismos de tamanho de fragmentos de restrição) foi utilizada com sucesso na separação das 10 espécies de Biomphalaria existentes no Brasil e outras espécies desse gênero oriundas de outras localidades da América do Sul (Argentina, Uruguai, Paraguai, Venezuela) e Cuba. Utilizndo a PCR-RFLP, foi possível separar: 1) as 10 espécies e uma subespécie de moluscos brasileiros do gênero Biomphalaria; 2) espécies similares encontradas no Brasil, Argentina e Uruguai tais como: B. kuhniana, B. intermedia, B. straminea, B. tenagophila, B. occidentalis, B. t. guaibensis, B. oligoza, B. peregrina e B. orbignyi; 3) populações de B. havanensis de Cuba, B. obstructa obtidas de Cuba e República Dominicana e Isla del Carmem, México e, 4) caracterizar populações de B. prona obtidas do lago de Valência e dos arredores deste (Venezuela). Essa técnica mostrou-se uma ferramenta taxonômica importante, confirmando, na maioria dos casos, a sistemática morfológica clássica reforçando as relações de similaridade entre espécies do gênero Biomphalaria pela criação do complexo B. tenagophila e demonstrando a alta similaridade genética entre B. peregrina e B. oligoza. A análise das seqüências da região espaçadora interna ITS2 permitiu estimar as relações filogenéticas das espécies brasileiras do gênero Biomphalaria. Foram utilizados 3 diferentes métodos de inferência filogenética (neighbor-joining, parcimônia e maximum likelihood). Nossos resultados mostraram que a região ITS1 é muito variável e que ITS2 contém marcadores moleculares apropriados para a determinação das relações filogenéticas entre as espécies de Biomphalaria. As árvores filogenéticas obtidas com estes métodos apoiaram a sistemática atual baseada em dados morfológicos. Neste estudo moluscos do gênero Helisoma foram usados como outgroup.

Identificação específica e filogenia de moluscos neotropicais do gênero Biomphalaria através de técnicas de biologia molecular

A identificação morfológica dos moluscos do gênero Biomphalaria é dificultada, principalmente, pela semelhança e extensa variação intraespecífica observada nos caracteres morfológicos utilizados na identificação clássica desses moluscos. Com o objetivo de contornar esses problemas, introduzimos metodologias moleculares como ferramentas adicionais à identificação morfológica. A análise de polimorfismo de seqüência da região espaçadora interna (ITS1 + 5.8S + ITS2) do gene codificador do RNAr de Biomphalaria, amplificada através da PCR (reação em cadeia da polimerase) e posterior digestão com enzimas de restrição (RFLP-polimorfismos de tamanho de fragmentos de restrição) foi utilizada com sucesso na separação das 10 espécies de Biomphalaria existentes no Brasil e outras espécies desse gênero oriundas de outras localidades da América do Sul (Argentina, Uruguai, Paraguai, Venezuela) e Cuba. Utilizndo a PCR-RFLP, foi possível separar: 1) as 10 espécies e uma subespécie de moluscos brasileiros do gênero Biomphalaria; 2) espécies similares encontradas no Brasil, Argentina e Uruguai tais como: B. kuhniana, B. intermedia, B. straminea, B. tenagophila, B. occidentalis, B. t. guaibensis, B. oligoza, B. peregrina e B. orbignyi; 3)populações de B. havanensis de Cuba, B. obstructa obtidas de Cuba e República Dominicana e Isla del Carmem, México e, 4) caracterizar populações de B. prona obtidas do lago de Valência e dos arredores deste (Venezuela). Essa técnica mostrou-se uma ferramenta taxonômica importante, confirmando, na maioria dos casos, a sistemática morfológica clássica reforçando as relações de similaridade entre espécies do gênero Biomphalaria pela criação do complexo B. tenagophila e demonstrando a alta similaridade genética entre B. peregrina e B. oligoza. A análise das seqüências da região espaçadora interna ITS2 permitiu estimar as relações filogenéticas das espécies brasileiras do gênero Biomphalaria.

Foram utilizados 3 diferentes métodos de inferência filogenética (neighbor-joining, parcimônia e maximum likelihood). Nossos resultados mostraram que a região ITS1 é muito variável e que ITS2 contém marcadores moleculares apropriados para a determinação das relações filogenéticas entre as espécies de Biomphalaria. As árvores filogenéticas obtidas com estes métodos apoiaram a sistemática atual baseada em dados morfológicos. Neste estudo moluscos do gênero Helisoma foram usados como outgroup.