Amplified fragment length polymorphism (AFLP) reveals species-specific markers in the Daphnia galeata–hyalina species complex Massimiliano Gili 1,2 , Michael T. Monaghan 1,3 & Piet Spaak 1, * 1 Department of Limnology, EAWAG, U ¨ berlandstrasse 133, Postfach 611, 8600 Du ¨bendorf, Switzerland 2 Present address: Via Stazione 78, 10060 None (Torino), Italy 3 Present address: Molecular Systematics Laboratory, Department of Entomology, The Natural History Museum, Cromwell Road, London SW7 5BD, UK (*Author for correspondence: Tel.: +41-1-823-5617; Fax: +41-1-823-5315; E-mail: spaak@eawag.ch) Key words: hybridization, molecular marker, backcrossing, introgression, Daphnia Abstract Daphnia often occur in species complexes that consist of two or more co-occurring species and their hybrids. Hybrid individuals are often capable of sexual reproduction and so backcrossing with introgres- sion occurs. To better understand hybridization and backcrossing frequency, we sought to develop PCR- based, species-specific markers in the Daphnia galeata–hyalina species complex using amplified fragment length polymorphism (AFLP). This technique produces large numbers of reproducible markers for assessing diversity across the nuclear genome and provides several advantages over mtDNA and micro- satellite approaches. We examined 28 clones of D. galeata, D. hyalina, and their hybrids isolated from Lake Constance on the Swiss-German border. Using a single AFLP primer combination we found five potential species-specific markers, defined as bands that occurred in >80% of one parental species and <20% of the other. Two bands appeared to be co-dominant and were present (homozygous) in D. galeata, absent in D. hyalina, and heterozygous in the hybrid. We conclude AFLP could provide enough PCR-based, species- specific markers to identify species, hybrids, and backcrosses from even small amounts of tissue (i.e. resting eggs). Introduction Determining the Daphnia species composition of European lakes is not always an easy task. Sibling species typically co-occur and often hybridize (Schwenk & Spaak, 1995). Although attempts have been made to distinguish taxa morphologically (Flo¨ßner, 1993; Gießler, 2001), it remains difficult for non-specialists to quantify species and hybrid frequencies. With the help of genetic techniques (allozymes), Wolf & Mort (1986) and Wolf (1987) found that certain Got (renamed to AAT ) alleles were species-specific and can be used to identify hybrids and parental species within the Daphnia galeata–hyalina–cucullata complex. Giebler (1997) identified AO as another diagnostic locus, provid- ing the possibility to distinguish backcrosses. Fur- ther studies have found that hybridization and backcrossing are common events in natural popu- lations (Spaak, 1996; Schwenk, 1997; Schwenk et al., 1998). All studies published to date show evidence for backcrossing within the Daphnia galeata–hyalina–cucullata species complex, but with only two species-specific markers it remains difficult to identify individual backcrosses (but see Jankowski, 2002; Spaak et al., 2004). Schwenk (1993) found species-specific mtDNA restriction sites, and since mtDNA is inherited maternally it can be used to investigate which species served as the maternal parent of a hybrid Hydrobiologia 526: 63–71, 2004. J. Pijanowska, P. Dawidowicz, A. Jachner & K. Szeroczyn ´ska (eds), Cladocera. Ó 2004 Kluwer Academic Publishers. Printed in the Netherlands. 63