1 Centro de Biologia Ambiental/Departamento de Zoologia e Antropologia, Faculdade de Cieˆncias, Universidade de Lisboa, Lisboa, Portugal; 2 A ´ rea de Biologı´a Animal, Departamento de Zoologı´a y Antropologı´a Fı´sica, Universidad de Murcia, Murcia, Spain Chromosome evolution in the genus Cicindela: physical mapping and activity of rDNA loci in the tiger beetle species Cicindela littoralis and C. flexuosa S.J.R. Proenc ¸a 1 and J. Galia ´n 2 Abstract Cicindela littoralis and Cicindela flexuosa were analysed at population level to determine the localization and activity of ribosomal genes. Fluorescence in situ hybridization (FISH), using a PCR-amplified 18S rDNA fragment as a probe, revealed the presence of polymorphism regarding the number of chromosomes with ribosomal genes as well as their localization within the genome. Nine populations of C. littoralis showedaconsistentpatternoftwolocilocatedinanautosomalpair(activeduringspermatogenesisasshownbysilverstaining)andonelocus locatedinoneofthemultipleXchromosomes(silentduringspermatogenesis),whereasindividualsfromthepopulationofPuntaEntinasshowed onlysignalsintheautosomalpair,lackingtheheterosomallocus.In C. flexuosa,twopatternswerealsoobserved.Ninepopulationsshowedtwo lociinanautosomalpairwhereasindividualsfromthepopulationofSanPedrodelPinatarshowedthetwolocilocatedintheheterosomes(one of the multiple Xs and in the Y). The hypothesis that these two different populations may reflect a status of well-differentiated phylogenetic entities is not supported for C. littoralis after the phylogenetic analysis of a fragment of the cytochrome oxidase I gene. Key words: Cicindela – Coleoptera – in situ hybridization – multiple sex chromosomes – physical mapping – rDNA – NORs Introduction Ribosomal DNA (rDNA) genes (18S, 5.8S and 28S) are clustered together in eukaryotic genomes in replication units repeated 100 or 1000 times and separated by intergenic non- transcribedspacers(HillisandDixon1991).Knowledgeofthe number and chromosomal localization of these highly repet- itive and conserved rDNA clusters via fluorescence in situ hybridization (FISH) is useful for both the construction of physicalmapsforcomparativegenomicsandphylogeneticand evolutionarystudies.Mostofthesestudiesinbeetleshavebeen concentrated on the families Carabidae (reviewed in Sa´nchez- Gea et al. 2000), Tenebrionidae and Chrysomelidae (reviewed in Petitpierre 1996). Tigerbeetlesofthegenus Cicindela arecharacterizedbythe presence of a multiple sex chromosome system (see Serrano and Galia´n 1998 for a summary list and references therein) thatformsanon-chiasmaticmultivalent.In Cicindela hybrida, the members of this multivalent group were shown to be connected by telomeric proteins during meiosis (Giers 1977). This system was found in most species of the genus Cicindela s.l, and is considered ancestral for the genus. Galia´n et al. (2002) postulated a single origin of the multiple sex chromo- some system, in a common ancestor to Collyrinae and Cicindelini. The generalized karyotype in the genus Cicindela is made up of 9–11 autosomal pairs plus a sex chromosome mechanism of the X n Y (males)/X n X n (females) type, where n variesbetween2and4(SerranoandGalia´n1998;Galia´nand Hudson 1999). However, single systems representing secon- darylosshavebeendescribedin Cylindera germanica (XY/XX) (Giers 1977; Galia´n et al. 2002) and in Cylindera paludosa (X0/XX) (Serrano et al. 1986). Silver staining of nuclear organizing regions (NORs) and FISH with a ribosomal probe have been carried out to study the chromosomal localization and activity of ribosomal DNA genes in Paleartic and Australian species of tiger beetles (Galia´n et al. 1995; Galia´n and Hudson 1999). FISH differs from silver staining in that it reveals the actual number of chromosomescarryingNORswhereassilverstainingindicates whichoftheseareactiveinaparticularstageofthecellcycle. Cicindela littoralis and Cicindela flexuosa, two species belonging to the tribe Cicindelini, have already been studied regarding the chromosome number and the sex chromosome mechanism (Giers 1977; Gue´nin 1952; Galia´n et al. 1990). Both species have nine pairs of autosomes and a sex chromosome system of the type X 1 X 2 X 3 Y in males and X 1 X 1 X 2 X 2 X 3 X 3 in females; that is a diploid value of 2n ¼ 22 inmalesand2n ¼ 24 in females. In this paper, 10 populations of each species covering the Iberian Peninsula (a population of C. littoralis from Morocco was also included) were analysed to determine the pattern of rDNAlocalization.SilverstainingofNORswascarriedoutin order to analyse the pattern of activity of the nucleolus through the cell cycle and to differentiate active from silent rDNA clusters. A fragment of 800 bp of the mitochondrial cytochromeoxidaseIgene(COI)wassequencedin C. littoralis to test the geographic grouping of its populations. Materials and methods Material Adult tiger beetles of both species were collected in different localities of Spain and Portugal (Table 1). The specimens were identified by the authors and deposited in the collection of the Department of Zoology and Anthropology, Univer- sity of Lisbon, and in the Department of Animal Biology, University of Murcia. Chromosome preparations Testes and ovaries from adult beetles were used to obtain mitotic or meiotic chromosomes and nuclei. Gonads were dissected from beetles anaesthetized with ethyl-acetate, hypotonically treated in distilled waterandfixedusingfreshethanol–aceticacidsolution(3:1)for1h, with several changes of the fixative solution during the next day and werekeptat )20°Cuntilstudied.Smallsectionsofthegonads(1mm) weresquashedonaslidein70%aceticacid;thecoverslipwasremoved J. Zool. Syst. Evol. Research 41 (2003) 227–232 Ó 2003 Blackwell Verlag, Berlin ISSN 0947–5745 Received on 23 July 2002 U.S. Copyright Clearance Center Code Statement: 0947–5745/03/4104–0227$15.00/0 www.blackwell.de/synergy