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

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.


Introduction
Cladocerans are important members of freshwater communities, both planktonic and benthic.( Elmoor-Loureiro, 1997).Some species make good indicators of the trophic state of the water bodies in which they occur (Paranaguá et al., 2005) and some genera (eg.Daphnia) have been used as models for ecological investigations, as test-organisms in ecotoxicological studies and more recently in ecological genetics.So far, 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).The morphological taxonomy of many species of Cladocera is a complex matter, considering that only small differences separate some species from others.Besides, different morphological forms from the same species of Ceriodaphnia could be related to the presence of predators (Rietzler et al., 2008).However, 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.Nowadays, 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).Ribosomal RNA (rRNA) genes have been widely used as molecular markers for taxonomic purposes.The spacer regions of the repeat unit favour fixation at a higher rate (since they are submitted to a neutral selective pressure) than the gene regions.Therefore 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).
A cladoceran genus of the family Daphnidae possessing morphologically similar species, which hampers the systematic biology of the group, is Ceriodaphnia (Berner, 1987;Elmoor-Loureiro, 1997).In the present study, two species of Ceriodaphnia were investigated: Ceriodaphnia dubia Richard, 1894, a species considered exotic, in Brazil, which was introduced as a test organism for ecotoxicological investigations (now already colonizing many water bodies) and the native species Ceriodaphnia silvestrii Daday, 1902, widely distributed in Brazilian freshwaters.These species are only distinguishable by details on the proximal pecten of the postabdominal claw, and even among specialists there is uncertainty in telling them apart (Elmoor-Loureiro, 1997).The aim of this study was, therefore, to search for molecular markers in order to genetically distinguish the native species C. silvestrii (both laboratory cultured clones and specimens found in the Furnas Hydroelectricity Power Plant Reservoir) from the exotic species C. dubia (laboratory grown).

Material and Methods
Zooplankton samples were collected in the Furnas Hydroelectricity Power Plant Reservoir by horizontal trawls with a 40 µm mesh zooplankton net.Samples were preserved in 50% ethanol to facilitate identification and isolation of individuals under the stereomicroscope in view of their rapid movements when alive.Subsequently, specimens were checked taxonomically by optical microscopy.Cladoceran DNA extraction was carried out with the QIAamp DNA Micro Kit (Qiagen), using a bulk of 150 individuals.The animals were pretreated in order to break down their carapaces: after adding the kit lysing buffer, the material was crushed.After this step, the kit protocol was followed.The polymerisation chain reaction (PCR) was used to amplify the ITS1-5.8S-ITS2region, in order to search for molecular markers that could discriminate both species.The primers required for the reaction were designed based on rRNA gene sequences found in GenBank: namely DUBIA 181 -5´GGAAGTAAAAGTCGTAACAAGG3´ (corresponding to the 3' end of C. dubia 18S/SSU rRNA gene) and DUBIA 28 -5´CGAGGTTCGGTGCTGGGCTCATCC3´ (complementary to the 5' end of the 28S/LSU rRNA gene).PCR reactions were performed using 100 ng of genomic DNA, 100 mM Tris-HCl, 500 mM KCl, 0.8% Nonidet P40, 1.5 mM MgCl 2 , 100 µM dNTPs, 50 pmol each primer and 2 units of Taq Recombinant DNA polymerase (Fermentas, Life Science).The amplification cycles were: 94 °C for 5 minutes; 30 cycles of 94 °C for 1 minute, 57 °C for 1 minute, 72 °C for 2 minutes; and a final extension at 72 °C for 10 minutes.After sizefractioning of products in agarose gels, each product was purified (Wizard ® SV gel and PCR clean-up system -Promega) and cloned into pGEM-T Easy Vector Kit (Promega), which has a plasmid of 3015 bp as cloning vector and a cleavage site for each of the restriction enzymes used in the analysis (in the linker), including two EcoRI sites flanking the PCR product cloning site.Braz.J. Biol., 2010, vol. 70, no. 1, p. 121-124 effective for the DNA extraction from cladocerans with sufficient quality and enough amplification in the PCR reaction to find molecular markers for taxonomic purposes.The present study is the first succeeding to isolate and characterise the complete ITS1-5.8S-ITS2region of the rRNA genes of cladocerans of the Ceriodaphnia genus, showing that its size is similar to that found in several species of the Daphnia genus, which is approximately 1900 bp (Taylor, Sprenger and Ishida, 2005).By comparing this region it is possible to assert that the species Ceriodaphnia silvestrii and Ceriodaphnia dubia, which are morphologically very similar, are actually two genetically distinct species.Additionally, the same pattern of restriction was found for the individuals of the species C. silvestrii analysed (both from lab cultures and from the Furnas Reservoir), corroborating the hypothesis that all of them belonged to that species.
It can therefore be concluded that the region ITS1-5.8S-ITS2 is a good molecular marker for differentiating the species of Ceriodaphnia studied, considering that it includes both conserved regions (such as the 5.8S region), and less conserved regions such as the ITS-1 and ITS-2 spacers, facilitating the task of distinguishing species that are morphologically very similar.This methodology might be very useful in determining the distribution of the exotic species C. dubia in Brazilian freshwater, particularly when C. dubia occurs in the absence of C. silvestrii, and it is not possible to make a ready morphological comparison of the two, a particularly difficult task for ecologists who are not taxonomy specialists.

Figure 1
Figure 1.a) General view; b) Proximal pecten of the postabdominal claw of Ceriodaphnia dubia; c) General view and d) Proximal pecten of the postabdominal claws of Ceriodaphnia silvestrii.