Cytogenetic analysis of Anaecypris hispanica and its relationship with the paternal ancestor of the diploid-polyploid Squalius alburnoides complex Marta Gromicho, Maria Manuela Coelho, Maria Judite Alves, and Maria Joa ˜ o Collares-Pereira Abstract: The karyotype of the endangered fish Anaecypris hispanica was revisited using advanced cytogenetic techniques to elucidate its putative relationship with the paternal ancestor of the hybrid complex Squalius alburnoides and to clarify some of the recently described cytogenetic patterns of the complex. The results of chromomycin A 3 and Ag staining, as well as fluorescent in situ hybridization with 28S and 5S rDNA and the (TTAGGG)n telomeric probes, were compared with the patterns observed in specimens of the all-male nonhybrid lineage of S. alburnoides complex, which is considered to reconstitute the nuclear genome of the probably extinct paternal ancestor. Several cytogenetic features observed in A. hispanica specimens were indeed shared by S. alburnoides nuclear nonhybrid males, supporting the hypothesis of a close evolutionary link between A. hispanica and the paternal ancestor of the complex. The genomic rearrangements in- volving 28S rDNA sites previously described in the S. alburnoides complex and in its maternal ancestor (S. pyrenaicus) were not detected in A. hispanica; they are, therefore, probably due to mechanisms related to hybridization and poly- ploidy. Key words: hybridization, polyploidization, CMA3 and Ag staining, 28S and 5S rDNA, telomeric sequence, Cyprinidae. Re ´sume ´: Le caryotype du poisson menace ´ Anaecypris hispanica ae ´te ´ re ´examine ´a ` l’aide de techniques cytoge ´ne ´tiques avance ´es afin d’e ´lucider sa possible relation avec l’ance ˆtre paternel du complexe hybride Squalius alburnoides et afin de clarifier certains des motifs cytoge ´ne ´tiques re ´cemment de ´crits chez ce complexe. Les re ´sultats de colorations au CMA 3 et a ` l’argent, de me ˆme que des hybridations in situ en fluorescence (FISH) avec les sondes d’ADNr 28S et 5S ainsi qu’avec la sonde te ´lome ´rique (TTAGGG) n , ont e ´te ´ compare ´s avec les motifs observe ´s chez des spe ´cimens appartenant au lignage non-hybride exclusivement ma ˆle du complexe S. alburnoides, lequel est conside ´re ´ reconstituer le ge ´nome nucle ´aire de l’espe `ce paternelle qu’on soupc ¸onne disparue. Plusieurs particularite ´s cytoge ´ne ´tiques observe ´es chez les spe ´cimens A. his- panica e ´taient en effet pre ´sentes chez les S. alburnoides ma ˆles a ` ge ´nome nucle ´aire non-hybride, ce qui supporte l’hypo- the `se d’une grande proximite ´e ´volutive entre A. hispanica et l’ance ˆtre paternel du complexe. Les re ´arrangements ge ´nomiques impliquant l’ADNr 28S, de ´crits ante ´rieurement chez le complexe S. alburnoides et chez son ance ˆtre maternel (S. pyrenaicus), n’ont pas e ´te ´ de ´tecte ´s chez A. hispanica. Ceux-ci sont ainsi vraisemblablement dus a ` des me ´canismes lie ´s a ` l’hybridation et la polyploı ¨die. Mots cle ´s : hybridation, polyploı ¨disation, coloration au CMA3 et a ` l’argent, ADNr 28S et 5S, se ´quence te ´lome ´rique, cypri- nide ´s. [Traduit par la Re ´daction] Introduction The Squalius alburnoides fish complex originated through interspecific crosses between Squalius pyrenaicus (P ge- nome) females and males of a probably extinct species (A genome) (Alves et al. 2001). The complex contains 2 dis- tinct forms in the southern Iberian drainages. The most com- mon comprises hybrid females and males with diploid (PA, 2n = 50), triploid (PAA and PPA, 3n = 75), and tetraploid (PPAA, PAAA, and PPPA, 4n = 100) genomes. The other form is a diploid nuclear nonhybrid all-male lineage (AA) also containing pyrenaicus-type mtDNA; it was therefore hypothesized to have been reconstituted inside the complex by triploid hybrid females PAA (Fig. 1). The all-male AA lineage permitted the preservation of the paternal ancestor Received 7 March 2006. Accepted 25 September 2006. Published on the NRC Research Press Web site at http://genome.nrc.ca on 23 February 2007. Corresponding Editor: W. Traut. M. Gromicho, M.M. Coelho, and M.J. Collares-Pereira. 1 Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Cie ˆncias, Universidade de Lisboa, Campo Grande C2 – Piso 3, 1749-016 Lisboa, Portugal. M.J. Alves. Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Cie ˆncias, Universidade de Lisboa, Campo Grande C2 – Piso 3, 1749-016 Lisboa, Portugal; Universidade de Lisboa, Museu Nacional de Histo ´ria Natural, Museu Bocage, Rua da Escola Polite ´cnica 58, 1269–102 Lisboa, Portugal. 1 Corresponding author (e-mail: mjpereira@fc.ul.pt). 1621 Genome 49: 1621–1627 (2006) doi:10.1139/G06-121 # 2006 NRC Canada