Evaluation of the 18S rRNA clone library approach to study the diversity of the macroeukaryotic leaf-epiphytic community of the seagrass Posidonia oceanica (L.) Delile

Abstract

The sequence comparisons among genes codifying for the RNA component of the small ribosomal subunit (16S rRNA or 18S rRNA) in cellular organisms have been largely used to reconstruct their phylogenies, and hence the identification of taxa by means of a molecular approach. Furthermore, the direct DNA isolation from environmental samples and the PCR amplification of the pool of rRNA genes with the subsequent cloning and sequencing have opened the door to the description of naturally occurring microbial communities independently from any culturing technique or morphological identification. These studies have unveiled an enormous hidden diversity in a wide variety of microbial communities. Our main objective was to evaluate the usefulness of the 18S rRNA gene clone libraries to describe the structure of the macroeukaryotic leaf-epiphytic assemblage of the seagrass Posidonia oceanica, and monitor the changes occurring in different stages of its seasonal succession (winter, spring and summer). To that end, we compared the results of these libraries with those provided by classical microscopy techniques. Among both approaches, the screening of clone libraries rendered the highest number of distinct units named operational phylogenetic units. However, diversity estimates provided by both methods were comparable and rendered the highest Shannon Diversity Index (H′) at the end of the succession. The major discrepancies were on the different occurrence of some groups. For example, macroalgae were the most frequent epiphytes counted by microscopy, whereas metazoa (specially, bryozoa) dominated the clone libraries. Altogether the results indicate that clone libraries constitute an excellent complementary approach to classical microscopy methods. To the best of our knowledge, this is the first attempt to describe a marine macroeukaryotic community using a molecular approach such as the analysis of 18S rRNA gene clone libraries.