© 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2017, 120, 626–636 626 Biological Journal of the Linnean Society , 2017, 120, 626–636. With 2 figures. Temporal frames of 45S rDNA site-number variation in diploid plant lineages: lessons from the rock rose genus Cistus (Cistaceae) CHIARA TOTTA 1 , MARCELA ROSATO 2 , PABLO FERRER-GALLEGO 3 , FERNANDO LUCCHESE 1 and JOSEP A. ROSSELL  O 2,4 * 1 Universit  a degli Studi Roma Tre, Viale G. Marconi 446, 00146, Rome, Italy 2 Jard ın Bot  anico-ICBiBE-Unidad Asociada CSIC, Universidad de Valencia, c/Quart 80, E46008, Valencia, Spain 3 CIEF, Servicio de Vida Silvestre, Generalitat Valenciana, Avda. Comarques del Pa ıs Valenci  a 114, E46930, Valencia, Spain 4 Carl Faust Fdn., PO Box 112, E17300, Blanes, Spain Received 5 August 2016; revised 30 August 2016; accepted for publication 30 August 2016 The perception that the turnover of 45S rDNA site number in plants is highly dynamic pervades the literature on rDNA evolution. However, most reported evidences come from the study of polyploid systems and from crop species subjected to intense agronomic selection. In sharp contrast with polyploids, the evolutionary patterns of rDNA loci number in predominantly diploid lineages have received less attention. Most studies on rDNA loci changes lack explicit temporal frames, and hence their dynamics could not be assessed. Here, we assess the temporal patterns of rDNA site evolution in Cistus, an entirely diploid lineage. We assessed the number and chromosomal position of 45S rDNA loci in Cistus species using fluorescence in situ hybridization (FISH) and Ag- nucleolus organizing regions (Ag-NOR) staining. Maximum likelihood and maximum parsimony reconstructions of the ancestral state of the 45S rDNA locus number were inferred onto a dated phylogeny. 45S rDNA locus number in Cistus ranged from one to four. Maximum likelihood and maximum parsimony reconstructions suggested that the two-loci state was the ancestral condition in Cistaceae, including the sister genera Tuberaria and Cistus. The most likely basal number of rDNA loci (two) has been maintained from the hypothesized ancient splitting events between Fumana and the remaining Cistaceae lineages in the Oligocene to most of the recent clades of Cistus diversified in the Middle Pleistocene. Our results support the view that evolutionary stasis regarding the number of 45S rDNA loci have been prevalent in several Cistus lineages and close relatives along their evolutionary history. It is suggested that conservation in rDNA site number likely occurred along more than 25 Mya of plant evolution, leading support to hypothesize that rDNA stasis in site number may have been neglected and underestimated in plant evolution at the diploid level. © 2016 The Linnean Society of London, Biological Journal of the Linnean Society, 2016, 00, 000–000. KEYWORDS: 45S rDNA – Cistaceae – Cistus – diploid lineages – fluorescent in situ hybridization – Fumana – Tuberaria. INTRODUCTION Genes encoding ribosomal RNA (rDNA) are universal constituents of cell genomes and are essential for organismal growth and integrity, since their prod- ucts form the backbones of cytoplasmic, plastidial, and mitochondrial ribosomes (Hillis & Dixon, 1991). In contrast to the single or low-copy number of rDNA genes present in the plastidial and mitochondrial genomes, the nuclear genome harbours hundreds to several thousand copies of each ribosomal species (18S, 5.8S, 25S/26S, 5S) that are usually arranged in distinct arrays of tandemly repeated units (Srivas- tava & Schlessinger, 1991). Although rDNA is the most abundant gene family and occupies a large fraction of the nuclear genome, *Corresponding author. Current address: Jard  ın Bot anico- ICBiBE-Unidad Asociada CSIC, Universidad de Valencia, c/Quart 80, E46008 Valencia, Spain. E-mail: rossello@uv.es 2017, 120, 626–636. Downloaded from https://academic.oup.com/biolinnean/article/120/3/626/3055996 by guest on 24 February 2022