Molecular Ecology Notes (2006) 6, 915–917 doi: 10.1111/j.1471-8286.2006.01398.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd PRIMER NOTE Development and localization of microsatellite markers for the sibling species Chironomus riparius and Chironomus piger (Diptera: Chironomidae) CARSTEN NOWAK,* THOMAS HANKELN,†‡ ERWIN R. SCHMIDT†‡ and KLAUS SCHWENK* *Department of Ecology and Evolution, J. W. Goethe-Universität, Siesmayerstr. 70, D-60054 Frankfurt am Main, Germany, †Institut für Molekulargenetik, Gentechnologische Sicherheitsforschung und Beratung, Johannes Gutenberg-Universität Mainz, Mainz, Germany, ‡ GENterprise GmbH, J.-J. Becherweg 32, D-55099 Mainz, Germany Abstract Five variable microsatellite loci are reported for the nonbiting midge species Chironomus riparius and Chironomus piger. All loci show considerable intraspecific variation and species- specific alleles, which allow to discriminate among the two closely related species and their interspecific hybrids, and to estimate genetic diversity within and between populations. Additionally, the loci were localized on C. riparius polytene chromosomes to verify their single copy status and investigate possible chromosomal linkage. The described markers are used in different studies with regard to population and ecological genetics and evolutionary ecotoxicology of Chironomus. Keywords: Chironomids, Chironomus piger, Chironomus riparius, microsatellite markers, sibling species Received 22 February 2006; revision accepted 17 March 2006 Chironomids are a worldwide-distributed family of nemat- ocerans, which occupy nearly all kinds of freshwater environ- ments (Armitage et al . 1995). They play a key role in many lake and river systems because of their high abundance and their detritus consumption, and they represent an important food resource for many bird and fish species (Armitage et al . 1995). Although several studies investigated population dynamics within the Chironomidae, no suitable DNA-based population genetic markers for species of this family have been developed so far. One of the most widely distributed and frequent species, Chironomus riparius Meigen is used as a model organism in ecotoxicological sediment biotests (OECD 2004). In order to answer the question if anthropogenic pollution stress has consequences on the genetic diversity of Chironomus populations in the laboratory and the field, we developed five microsatellite markers for this species. These markers were tested for their applicability to its sister taxon Chironomus piger Strenzke, which is morphologically highly similar to C. riparius and has been shown to form interspecific hybrids with the latter species (Hägele 1999). Furthermore, the loci were located on the salivary gland chromosomes of the species via in situ hybridization in order to test for single copy status and physical independence. For the construction of a genomic DNA library, we extracted DNA from 100 individuals of a C. riparius culture sampled in Bochum (Germany). After shearing of the genomic DNA by nebulization, 0.5 –1.5 kb sized fragments were separated electrophoretically, electroeluted and purified. The fragments were ligated into pUC 19 vector plasmids cut with Sma I and then transformed into DA10B Escherichia coli host cells via electroporation. To identify fragments containing typical microsatellite DNA sequence motifs, we picked 2700 insert-containing clones on gridded nitro- cellulose filters (Schleicher & Schuell, BA85) for colony filter hybridization (Grunstein & Hogness 1975). Clones were screened for microsatellites using six different radioactive labelled synthetic oligonucleotide probes [(CA 15 , (GA) 15 , (AAT) 10 , (AAG) 10 , (ATG) 10 , (GATA) 6 ]. Fifty-five hybrid- izing clones were picked at random, and their inserts were amplified by polymerase chain reaction (PCR) using standard forward and reverse vector primers. Amplification products were column purified (QIAGEN), sequenced by dye ter- minator cycle sequencing using the ABI BigDye version 3.1 kit (Applied Biosystems) and separated on an ABI 3730 capillary sequencer. For genotyping microsatellite loci, Correspondence: Carsten Nowak, Fax: +4979824910; E-mail: c.nowak@zoology.uni-frankfurt.de