RESEARCH ARTICLE S. Papakostas Æ A. Triantafyllidis Æ I. Kappas T. J. Abatzopoulos The utility of the 16S gene in investigating cryptic speciation within the Brachionus plicatilis species complex Received: 21 January 2005 / Accepted: 30 March 2005 / Published online: 25 May 2005 Ó Springer-Verlag 2005 Abstract Recent reports indicate an extensive amount of molecular evolution separating cryptic taxa as well as significant population structure at a microgeo- graphical scale. Appropriate molecular markers are particularly suitable for distinguishing cryptic biologi- cal 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 mito- chondrial 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 mito- chondrial 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 an- cient separations. It has also exhibited maximum dis- criminatory power (100% success) between lineages during RFLP analysis. The 16S assayed region has proven especially informative and consistent in detect- ing, supporting and establishing the lineage status within the B. plicatilis species complex both from a phylogenetic perspective and as an identification tool. Introduction For many years, it has been a rather entrenched notion that the high dispersive potential of aquatic taxa (i.e. passive transport of their diapausing propagules via wind or waterfowl) was likely to result in their entire world populations being nearly panmictic (Mayr 1963). Consequently, zooplanktonic species have long been effectively regarded as geographically invariant due to high rates of gene flow. However, the development and application of molecular tools in the last 2 decades have revealed high levels of population subdivision at a mi- crogeographical scale (De Meester 1996; Hebert 1998). As a result, many aquatic invertebrates are now recog- nized as cryptic species complexes with a high degree of regional endemism (Colbourne et al. 1997; Serra et al. 1997; Hebert 1998; King and Hanner 1998; Taylor et al. 1998; Witt and Hebert 2000; Abatzopoulos et al. 2002a; Penton et al. 2004; Wellborn and Cothran 2004). Monogonont rotifers are a striking example of a group of organisms where research from a molecular ecological perspective has overturned long-standing views on patterns of genetic differentiation and adaptive divergence of continental aquatic invertebrates. Recent advances, mainly through application of powerful DNA assays, have considerably refined our knowledge on the evolutionary relationships among these zooplanktonic taxa. Rotifers of the class Monogononta comprise important components of fresh and brackish water eco- systems (Hutchinson 1967). Among monogononts, Brachionus plicatilis (Mu¨ller) is found at salinities be- tween 2 ppt and 97 ppt (see Lowe et al. 2005) and con- Communicated by O. Kinne, Oldendorf/Luhe S. Papakostas Æ A. Triantafyllidis Æ I. Kappas T. J. Abatzopoulos (&) Department of Genetics, Development and Molecular Biology, Aristotle University of Thessaloniki, 541 24, Thessaloniki, Greece E-mail: abatzop@bio.auth.gr Tel.: +30-2310-998301 Fax: +30-2310-998256 Marine Biology (2005) 147: 1129–1139 DOI 10.1007/s00227-005-0012-7