Molecular Ecology (2006) 15, 4487–4497 doi: 10.1111/j.1365-294X.2006.03100.x © 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd Blackwell Publishing Ltd Diversity and natural hybridization in a highly endemic species of Petunia (Solanaceae): a molecular and ecological analysis ALINE P. LORENZ-LEMKE,* GERALDO MÄDER,* VALÉRIA C. MUSCHNER,* JOÃO R. STEHMANN,† SANDRO L. BONATTO,‡ FRANCISCO M. SALZANO * and LORETA B. FREITAS * *Departamento de Genética, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Caixa Postal 15053, 91501-970 Porto Alegre, RS, Brazil, †Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Antônio Carlos 6627, 31270-110 Belo Horizonte, MG, Brazil, ‡Centro de Biologia Genômica e Molecular, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Ipiranga 6681, 90610-001 Porto Alegre, RS, Brazil Abstract Intrinsic reproductive barriers among the species of Petunia are weak and genetic isolation is obtained mainly by geographical separation and ecological diversification. The Serra do Sudeste region in the extreme south of Brazil is one of the centres of diversity of this genus and is characterized by the presence of species with different pollination syndromes. Petunia exserta is known only from four sandstone towers in a restricted area of this region (about 500 km 2 ) and is characterized by its differentiated habitat (shelters in the sandstone towers) and by its floral characteristics adapted to ornithophily. In towers where this species is sympatric with the sphingophilous Petunia axillaris, phylogenetically close to P. exserta, we found plants with intermediate floral morphology, suggesting hybridization between them. To test this hypothesis and to better understand its consequences we ana- lysed the sequences of the plastid trnH-psbA, trnS-trnG and psbB-psbH intergenic spacers in 121 individuals sampled all over the P. exserta distribution. The joint analysis of the three markers revealed 13 haplotypes and the network showed two main genetic clades, which probably represent the original gene pool of the two species in the region. In general, individuals of a given population presented the same haplotype, independently of phenotype, corroborating the hybridization hypothesis. Field observations suggest that hummingbirds are responsible for the interspecific gene flow. Analysis of molecular variance revealed high interpopulational diversity among the towers. The low gene flow between populations is possibly related to the autochoric seed dispersion system. Keywords: conservation, evolution, hybridization, Petunia axillaris, Petunia exserta, speciation Received 19 March 2006; revision received 14 June 2006; accepted 10 July 2006 Introduction Complex morphological and physiological characteristics in angiosperm flowers evolved to attract specific pollinators (pollination syndromes) as a means to increase their reproductive efficiency. Pollinators are important factors in the evolution of flower shapes and colours and may contribute to reproductive isolation via reduced gene flow by pollen and differential visitation (Hodges & Arnold 1994; Proctor et al. 1996). These evolutionary changes may occur by mutations in genes with major effects in the flower appearance, such as on genes that control pigments (Bradshaw & Schemske 2003). In Petunia Juss. (Solanaceae), an extensive diversification of the pollination syndromes occurred and melitophilous, sphingophilous and ornitho- philous species can be found. Known as garden petunias, this genus has a long history (since the 19th century) of artificial crossings, and the hybrids between Petunia axillaris (Lam.) Britton, Sterns & Poggenb. and Petunia integrifolia (Hook.) Schinz & Thell. are disseminated worldwide as ornamental plants (Petunia hybrida). Correspondence: Loreta B. Freitas, Fax: 55 51 3316–9823; E-mail: loreta.freitas@ufrgs.br