Comparative genomics and repetitive sequence divergence in the species of diploid Nicotiana section Alatae

SummaryCombining phylogenetic reconstructions of species relationships with comparative genomic approaches is apowerful way to decipher evolutionary events associated with genome divergence. Here, we reconstruct thehistory of karyotype and tandem repeat evolution in species of diploid Nicotiana section Alatae. By analysis ofplastid DNA, we resolved two clades with high bootstrap support, one containing N. alata, N. langsdorffii,N. forgetiana and N. bonariensis (called the n ¼ 9 group) and another containing N. plumbaginifolia andN. longiflora (called the n ¼ 10 group). Despite little plastid DNA sequence divergence, we observed, viafluorescent in situ hybridization, substantial chromosomal repatterning, including altered chromosomenumbers, structure and distribution of repeats. Effort was focussed on 35S and 5S nuclear ribosomal DNA(rDNA) and the HRS60 satellite family of tandem repeats comprising the elements HRS60, NP3R and NP4R. Wecompared divergence of these repeats in diploids and polyploids of Nicotiana. There are dramatic shifts in thedistribution of the satellite repeats and complete replacement of intergenic spacers (IGSs) of 35S rDNAassociated with divergence of the species in section Alatae. We suggest that sequence homogenization hasreplaced HRS60 family repeats at sub-telomeric regions, but that this process may not occur, or occurs moreslowly, when the repeats are found at intercalary locations. Sequence homogenization acts more rapidly (atleast two orders of magnitude) on 35S rDNA than 5S rDNA and sub-telomeric satellite sequences. This rapidrate of divergence is analogous to that found in polyploid species, and is therefore, in plants, not onlyassociated with polyploidy.Keywords: comparative genomics, DNA phylogenetics, Nicotiana, tandem repeats, karyotypes, divergence.IntroductionClassical cytogenetic studies have revealed that alteredchromosome numbers and sizes are associated with speci-ation (Levin, 2002). The best-described examples of karyo-type evolution associated with plant speciation involvepolyploidy. Studies have revealed that polyploidy is associ-ated with gene conversion, sequence loss, gain, amplifica-tion and reduction, and all known forms of chromosomaltranslocation and mutation (Leitch and Bennett, 1997; Levyand Feldman, 2002; Liu and Wendel, 2003; Rieseberg, 2001;Skalicka et al., 2003, 2005; Soltis et al., 2004; Wendel, 2000).However, there are rather fewer papers describing suchphenomena in a set of diploid species within a phylogeneticcontext. Linking phylogenetics with comparative genomicsprovides a powerful means to determine the histories anddynamics of chromosome and tandem repeat divergence.Here we examine chromosome and sequence divergence inthespeciesofNicotianasectionAlatae(Solanaceae)tobetterunderstand genome evolution in plants at the diploid level.In terms of both the number of species already analysed(15) and the proportion of the genus analysed (20%),Nicotiana is already one of the best known plant genera forcomparative genome analyses (Clarkson et al., 2005; Limet al.,2000b,2004a,b,2005;Murad et al.,2002). InNicotiana,we have an ideal model system in which to investigate