The Fiocruz-CMM collection contains taxonomic groups of medical or veterinary relevance to aid in the control of schistosomiasis, support research, and contribute to human-resources development. Considering the global mobilization, consolidation, institutionalization, and organization of biological collections, studies evaluating the taxonomic accuracy of such collections are broadly relevant (Godfray and Knapp 2004; Egler and Santos 2006). The morphological identification of Biomphalaria species is challenging (Paraense 1988; Paraense et al. 1992), and PCR-RFLP has been used to aid in species identification (Spatz et al. 1999; Vidigal et al. 2000a; Caldeira et al. 2000; Carvalho et al. 2004). Thus, both tools were used to the taxonomic accuracy of the Fiocruz-CMM collection.
Initially, 8,831 specimens from 1,398 collection points were selected for this study. Specimens from 333 collection points were unidentified, while specimens from 198 collection points had already received molecular confirmation. Thus, this study evaluated the species identifications of mollusks from 1,200 collection points deposited at Fiocruz-CMM.
Identification errors occurred mainly between B. peregrina and B. tenagophila (29.1%), due to the similarity of these species’ shell and reproductive organs (Paraense 1966a); and between B. tenagophila and B. glabrata (20.8%), due to the many identical traits of juvenile and sometimes adults B. glabrata and B. tenagophila specimens (Barbosa 1964, Paraense and Deslandes 1959). The frequencies of these errors reflect the proportions of these species in the Fiocruz-CMM. The identification of certain B. havanensis specimens as B. temascalensis and B. obstructa was adequate because Yong et al. (2001) and DeJong et al. (2001) have concluded that these three entities actually form a single species, making B. temascalensis and B. obstructa junior synonyms of B. havanensis.
Three groups were considered inconclusive: B. aff. straminea, B. tenagophila from Argentina, and B. peregrina. Paraense and Corrêa (1989) classified a population from Espinillar, Uruguay, as B. aff. straminea due to its similarity to B. straminea. Vidigal et al. (1998) obtained molecular profiles with two bands (470 and 310 bp) for B. straminea populations from the city of San Miguel and the provinces of Chaco and Corrientes, Argentina, and four bands (470, 310, 280, and 120 bp) for B. straminea populations from Brazil. In the present study, specimens from Corrientes, Argentina and Espinillar, Uruguay had profiles with two bands, agreeing with the observations of Vidigal et al. (1998), and were morphologically similar to B. straminea.
Specimens from Argentina that were morphologically identified as B. tenagophila showed a profile with three bands, similar to that reported by Vidigal et al. (1998) for populations from Chaco and Corrientes, Argentina, that were morphologically identified as B. tenagophila. However, LHMM has previously used a profile with two bands to characterize B. tenagophila (Vidigal et al. 2000a). Spatz et al. (1999) has observed that B. tenagophila from Argentina exhibits greater phylogenetic proximity to both B. occidentalis and B. t. guaibensis than to B. tenagophila from Brazil. Paraense (1961) found no morphological differences between B. tenagophila populations from Corrientes, Argentina (the type locality of the species) and Brazil. Paraense (1961) also confirmed that these populations were conspecific by performing controlled crosses.
Some specimens that were morphologically identified as B. peregrina showed the molecular profile of that species, while others showed that of B. oligoza when using Alu I to digest either ITS (Figure 2) or COI (data not shown). The morphological separation of B. peregrina and B. oligoza is primarily based on the number of prostatic diverticula, which ranges from zero to seven in B. oligoza (Paraense 1975) and from eight to twenty-two in B. peregrina (Paraense 1966b, 1975). However, the specimens that exhibited the molecular profile of B. oligoza in this study had 10 to 18 diverticula. According to Vidigal et al. (2000b), the phylogenetic positions of these two species are uncertain because B. oligoza specimens are always grouped together, but B. peregrina specimens are ultimately grouped with B. oligoza. Thus, the number of diverticula may not be a definitive trait for the separation of these species.
Further morphological and molecular studies and experimental crosses are needed to establish the phylogenetic relationships among the members of these three groups, specially the specimens from inconclusive group. The part of the Cytochrome c oxidase subunit I gene (cox1) will be sequenced to better understanding of the taxonomic status of these species. This region has been widely used in taxonomic studies an approach termed DNA barcode (Hebert et al. 2003).
Some specimens could not be identified due to tissue degradation resulting from the lack of periodic maintenance. This result highlights the importance of adequately maintaining biological collections (Egler and Santos 2006).
The results of this study confirm the relevance of molecular taxonomic techniques in evaluating and updating the species identifications of Biomphalaria specimens and the need to guarantee proper specimen preservation. Importantly, the consolidation of this collection and the performance of this study were made possible by funding from Fiocruz and the Minas Gerais Research Foundation (Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Fapemig), confirming the need for financial support to strengthen biological collections.