The utility of the 16S gene in investigating cryptic speciation within the Brachionus plicatilis species complex

Abstract

Recent reports indicate an extensive amount of molecular evolution separating cryptic taxa as well as significant population structure at a microgeographical scale. Appropriate molecular markers are particularly suitable for distinguishing cryptic biological species. In this study, we examine the phylogenetic utility of 16S rRNA in elucidating the evolutionary relationships within the recently described euryhaline Brachionus plicatilis species complex. In addition, we assess the applicability of this marker in the genetic identification and monitoring of rotifer populations. We have sequenced a 378-bp fragment of the mitochondrial 16S rRNA gene in laboratory reference strains, hatchery clones as well as collections from a wild population of the subsaline Lake Koroneia (Northern Greece). Also, restriction fragment length polymorphism (RFLP) analysis was performed with eight restriction endonucleases. Rotifer samples are distinguished into six genetically divergent lineages. Average sequence divergence between lineages is 0.1038. The evolutionary relationships and divergence time-scales revealed with the 16S sequence data are in agreement with previous analyses using different mitochondrial and nuclear markers. The 16S region appears to have several advantages over other regions of the genome regarding use of species-specific primers, ease of amplification from single specimens and undiluted informational content over both recent and more ancient separations. It has also exhibited maximum discriminatory power (100% success) between lineages during RFLP analysis. The 16S assayed region has proven especially informative and consistent in detecting, supporting and establishing the lineage status within the B. plicatilis species complex both from a phylogenetic perspective and as an identification tool.