Reverse taxonomy: an approach towards determining the diversity of meiobenthic organisms based on ribosomal RNA signature sequences

Abstract

Organisms living in or on the sediment layer of water bodies constitute the benthos fauna, which is known to harbour a large number of species of diverse taxonomic groups. The benthos plays a significant role in the nutrient cycle and it is, therefore, of high ecological relevance. Here, we have explored a DNA-taxonomic approach to access the meiobenthic organismic diversity, by focusing on obtaining signature sequences from a part of the large ribosomal subunit rRNA (28S), the D3-D5 region. To obtain a broad representation of taxa, benthos samples were taken from 12 lakes in Germany, representing different ecological conditions. In a first approach, we have extracted whole DNA from these samples, amplified the respective fragment by PCR, cloned the fragments and sequenced individual clones. However, we found a relatively large number of recombinant clones that must be considered PCR artefacts. In a second approach we have, therefore, directly sequenced PCR fragments that were obtained from DNA extracts of randomly picked individual organisms. In total, we have obtained 264 new unique sequences, which can be readily placed into taxon groups, based on phylogenetic comparison with currently available database sequences. The group with the highest taxon abundance were nematodes and protozoa, followed by chironomids. However, we find also that we have by far not exhausted the diversity of organisms in the samples. Still, our data provide a framework within which a meiobenthos DNA signature sequence database can be constructed, that will allow to develop the necessary techniques for studying taxon diversity in the context of ecological analysis. Since many taxa in our analysis are initially only identified via their signature sequences, but not yet their morphology, we propose to call this approach 'reverse taxonomy'.

Figure 1

Scheme of the universal eukaryotic rRNA gene unit and depiction of the primer locations used in this study. Ribosomal gene clusters (depicted in the middle) are arranged in tandem (depicted at the bottom). The D3–D5 region is depicted at the top. The primers 1274 and 1275 were used to amplify the D3 region, the primers 1274 and 689 were used to amplify the whole D3–D5 region.

Figure 2

Location of the Bavarian lakes from which samples were obtained. The acronyms are listed in table 1.

Figure 3

Divergence profile of the sequences in our analysis correlated with secondary structure. The divergence measures are based on similarity values provided by the Arb program, the naming of the helices and loops follows Hassouna et al. (1984). The depiction does not include indel differences, which can be substantial.

Figure 4

(a) The abundance of taxa in general taxonomic classes. The assignment to one of the classes is based on the phylogenetic analysis obtained in Arb (see Electronic Appendix). Gas, Gastrotricha; Tur, Turbellaria; Tar, Tardigrada; Ins, Insecta without chironomids; Ost, Ostracoda; Ano, Anomopoda; Aca, Acari; Rot, Rotifera; Har, Harpacticoida; Cyc, Cyclopoda; Chi, Chironomida; Pro, Protozoa; Nem, Nematoda. (b) Reoccurrence of taxa in different lakes. The dark grey parts refer to the fraction of sequences that were found in more than one lake in each taxon class. The numbers refer to the actual numbers.

Figure 5

Taxon representation in lakes. The numbers of different sequences in each taxon group are related to the lake in which they were found. It is evident that major differences exist with respect to taxon representation in the different lake samples. Taxon groups as in figure 4a, but Crustaceans (Cru) and insects are in single groups.

Figure 6

Graphical representation of the accumulation of new unique sequences with every lake sample added. The addition of lake samples was in a random order. The graphic shows that there is no sign of saturation of the diversity obtained. On average about 75% of the sequences of each new sample were new sequences.