The use of PCR-RFLP to genetically distinguish the morphologically close species: Ceriodaphnia dubia Richard, 1894 and Ceriodaphnia silvestrii Daday, 1902 (Crustacea Cladocera)

Mj Abreu,Mj Santos-Wisniewski,Tc Orlando,O Rocha

doi:10.1590/s1519-69842010000100016

Abstract

The cladocerans are important components of planktonic and benthic freshwater and good indicators of the trophic state of water bodies. The morphological taxonomy of many species of Cladocera is considered complex with minor differences separating some species. Nowadays, molecular techniques provide a powerful tool to identify and classify different taxonomical levels, using mainly ribosomal RNA genes (rRNA) as molecular markers. In the present work we performed PCR-RFLP to separate Ceriodaphnia dubia, an exotic species in Brazil and the native species Ceriodaphnia silvestrii, widely distributed in Brazilian freshwater. The RFLP analysis of the ITS1-5.8S-ITS2 region of rRNA genes showed to be different between C. dubia and C. silvestrii when using enzymes EcoRI, ApaI and SalI. Thus, the ITS1-5.8S-ITS2 region proved to be a useful molecular marker to differentiate the studied Ceriodaphnia species, which makes the task easier of telling apart species that are morphologically very similar. Also, this methodology might be interesting in determining the distribution of the exotic species C. dubia in Brazilian freshwaters, particularly in cases when C. dubia occurs in the absence of C. silvestrii, a particularly difficult task for ecologists who are not taxonomy specialists.

Highlights

Cladocerans are important members of freshwater communities, both planktonic and benthic. (Elmoor-Loureiro, 1997)
The internal transcribed spacers (ITS) regions are rather interesting for studies on taxonomy and identification, because the spacer sequences have revealed intraspecific variation when analysed by restriction fragments length polymorphism (RFLP), frequently both in sequence and size (Sollner-Webb and Mougey, 1991; Matioli, 2001)
The specimens of C. dubia and C. silvestrii used in the present study were distinguishable by details on the proximal pecten of the postabdominal claw as seen in Figure 1 The polymerisation chain reaction (PCR) reaction resulted in a product of approximately 1900 bp in size for the ITS1-5.8S-ITS2 region of the Ribosomal RNA (rRNA) genes of C. dubia and C. silvestrii, obtained from laboratory cultures and isolated from Furnas Reservoir (Figure 2)

Summary

Introduction

Cladocerans are important members of freshwater communities, both planktonic and benthic. (Elmoor-Loureiro, 1997). Daphnia) have been used as models for ecological investigations, as test-organisms in ecotoxicological studies and more recently in ecological genetics. Many details of the life cycle, gene flow and population genetics of cladocerans are known for only a few species (Fonseca and Rocha, 2004; Schwenk et al, 1998). Correct identification at the species level is very important when evaluating biodiversity at infraspecific level or estimating genetic variability of a population (Rocha; Güntzel, 1999). Even when the ecological characteristics of a species are well known, biogeographic studies can be impaired by problems with complex taxonomical affiliations. Recently-developed molecular techniques that aim to identify and classify organisms at various taxonomic levels, are a powerful aid to comparative analysis (Schwenk et al, 1998). The internal transcribed spacers (ITS) regions are rather interesting for studies on taxonomy and identification, because the spacer sequences have revealed intraspecific variation when analysed by RFLP, frequently both in sequence and size (Sollner-Webb and Mougey, 1991; Matioli, 2001)

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Results