Molecular Phylogenetics and Evolution 39 (2006) 276–281 www.elsevier.com/locate/ympev 1055-7903/$ - see front matter  2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2005.08.009 Short communication Molecular insights on the taxonomic position of the paternal ancestor of the Squalius alburnoides hybridogenetic complex J.I. Robalo ¤ , C. Sousa Santos, A. Levy, V.C. Almada Instituto Superior de Psicologia Aplicada, Unidade de Investigação em Eco-Etologia, Rua Jardim do Tabaco 34, 1149-041 Lisbon, Portugal Received 23 June 2005; revised 9 August 2005; accepted 22 August 2005 Available online 4 October 2005 1. Introduction Many hybrid Wsh lineages result from intrageneric crosses and are often asexual, being considered as “evolu- tionary dead-ends” (Vrijenhoek, 1998). This seems not to be true in the Squalius alburnoides (Iberian minnow) Steindachner 1866 complex. This complex, that seems to have originated in a cross between distant species, has a remarkable variability in ploidy level and genomic compo- sition, includes fertile Wsh of both sexes and some of its forms retain meiosis and recombination (Alves et al., 2001). The S. alburnoides complex probably originated from crosses between S. pyrenaicus females and males from an unknown species. The complex includes 2n D 50, 3n D 75, and 4n D 100 hybrid forms (reviewed in Alves et al., 2001) with varying proportions of the two parental genomes (denoted by P and A, corresponding to the S. pyrenaicus and paternal ancestor genomes, respectively). Reconsti- tuted diploid non-hybrids (AA genome) are also produced and are morphologically distinct from the diploid hybrid form of the complex (PA). These non-hybrid Wsh, normally males (females seem to be extremely rare), exhibit the nuclear genome of the paternal ancestor (AA) and S. pyre- naicus-like mtDNA. In the absence of an identiWed paternal species this suggests that they are reconstituted from the hybrids (Alves et al., 2002). The oogenesis of triploid females with PAA genomes frequently involves discarding the Squalius (P genome), followed by normal meiosis and recombination, generating A gametes. When these gametes fuse with the sperm of AA males, which also undergo nor- mal meioses, new AA male progeny is generated. In the absence of S. pyrenaicus, such as in the northern basins of Portugal, this complex seems to be maintained by crosses with males of S. carolitertii (CC) and by diploid hybrid males (CA), although the mtDNA found in S. alburnoides Wsh is S. pyrenaicus-like (Cunha et al., 2004; Pala and Coelho, 2005). The Leuciscini presently found in the Iberian Peninsula include mainly species of the genera Squalius, Chondrostoma, and Anaecypris. Studies based on allozymes showed that S. alburnoides did not originate from intrageneric crosses between Squalius species and also ruled out members of the genera Chondrostoma and Anaecypris as paternal ancestors (Alves et al., 1997; Carmona et al., 1997). Recent studies still unpublished (Gilles, Dowling, Alves, Coelho, and Collares- Pereira) using introns from two nuclear genes seem to sug- gest that perhaps A. hispanica-like individuals may represent the paternal ancestor of this complex. The aim of the present work was to investigate which of the genera considered is phylogenetically closest to the paternal species that originated this complex. This approach is based on the ampliWcation of a segment of the -actin nuclear gene from a number of genera closely related to S. pyrenaicus and from the non-hybrid males of S. alburnoides. The topology obtained with -actin was compared to relatively complete phylogenies of the Euro- pean cyprinids based on cyt b (e.g., Briolay et al., 1998; Gil- les et al., 1998; Zardoya and Doadrio, 1999) and with our own reconstruction using a set of species comparable to that used with -actin. 2. Methods Total genomic DNA was extracted from Wn clips pre- served in ethanol by an SDS/proteinase-k-based protocol (adapted from Sambrook et al., 1989). For the -actin gene a total of 1062 bp was ampliWed using the primers For–5'-ATGGATGATGAAATTGCC * Corresponding author. Fax: +351 218 860 954. E-mail address: jrobalo@ispa.pt (J.I. Robalo).