Two Chloroplast DNA Inversions Originated Simultaneously During the Early Evolution of the Sunflower Family (Asteraceae)

Two Chloroplast DNA Inversions Originated Simultaneously During the Early Evolution of the Sunflower Family (Asteraceae) Ki-Joong Kim,* Keung-Sun Choi,* and Robert K. Jansen *School of Life Sciences and Biotechnology, Korea University, Seoul, Korea; and Section of Integrative Biology and Institute of Cellular and Molecular Biology, University of Texas Introduction Chloroplast genome organization is highly conserved among land plants (Palmer 1991; Raubeson and Jansen 2005). Gene orders may sometimes be reversed by large inversions that are mediated by intramolecular recombination events (Ogihara, Terachi, and Sasakuma 1988; Hiratsuka et al. 1989). The low levels of homoplasy and the overall rarity of large inversions among land plant chloroplast genomes suggest that these types of rare genomic changes are very reliable phylogenetic markers (Raubeson and Jansen 2005). Several large inversions have proven to be useful phylogenetic markers in a number of land plant groups, including the three large flowering plant families: Asteraceae, Fabaceae, and Poaceae (Jansen and Palmer 1987a; Doyle et al. 1992, 1996). In the sunflower family (Asteraceae), Jansen and Palmer (1987a, 1987b) identified two major lineages based on the distribution of a 22-kb inversion. This ancient dichotomy in the family was later supported with morphological (Bremer 1987) and chloroplast DNA (cpDNA) sequence data (Kim et al. 1992; Kim and Jansen 1995). The Asteraceae is one of the largest flowering plant families with approximately 1,535 genera and 23,000 species (Bremer 1994). The family includes many economically important species such as sunflower, lettuce, and artichoke, as well as many ornamentals. The Asteraceae has been the subject of intensive phylogenetic analyses using both morphological (Karis, Källersjö, and Bremer 1992) and molecular data (Kim et al. 1992; Kim and Jansen 1995). As a result, intrafamilial relationships among the major clades are relatively well established (Bremer et al. 1992; Bremer 1994; Kim and Jansen 1995). However, the times of origin and diversification of major clades of Key words: chloroplast DNA inversion, nonparametric rate smoothing, molecular clock, Asteraceae. E-mail: . Mol. Biol. Evol. 22(9):1783–1792. 2005 doi:10.1093/molbev/msi174 Advance Access publication May 25, 2005 Ó The Author 2005. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: Asteraceae still remain controversial due in part to the uncertainty of the early fossil record. The previous report of a cpDNA inversion from Asteraceae is derived from gene mapping using Southern hybridization (Jansen and Palmer 1987a, 1987b). Here we further characterize the inversion based on DNA sequence data. In addition, we identify a new 3.3-kb inversion that is coincident with one end point of the large inversion. Comprehensive sequence comparisons among 56 species of Asteraceae and related families enable the identification of the end points of the two inversions. We also estimate the times of origin for the inversion events using molecular clocks based on sequences of subunit six of chloroplast nicotinamide adenine dinucleotide (phosphate)H, NAD(P)H dehydrogenase (ndhF) and a large subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (rbcL). Materials and Methods Sequence Determination and Gene Identification of Lactuca sativa Chloroplast Genome in the Inversion Regions Four cloned cpDNA fragments (7.7, 7.2, 7.1, and 6.7 kb, fig. 1) containing the inversion end points of the Lactuca sativa chloroplast genome (Jansen and Palmer 1987a) were subcloned into pBluescript II vector using a combination of the four restriction enzymes BamHI, ClaI, EcoRI, and HindIII. Vector-inserted cpDNA fragments were sequenced using the BigDye 3.0TM terminal cycle sequencing kit (Applied Biosystems, Foster City, Calif.) and an ABI 3700 sequencer. Sequences were assembled using Sequencher (version 4.1; Gene Codes Corporation, Ann Arbor, Mich.). Gene annotations and comparative sequence analyses were performed using Blast and open reading frame finder programs from National Center for Biotechnology Information and ClustalX (Thompson et al. 1997). Published chloroplast genome sequences of Nicotiana and Panax were used for comparative analyses (Shinozaki et al. 1986; Kim and Lee 2004). The locations and secondary structures of trn genes were estimated using tRNAscan-SE The chloroplast DNA (cpDNA) inversion in the Asteraceae has been cited as a classic example of using genomic rearrangements for defining major lineages of plants. We further characterize cpDNA inversions in the Asteraceae using extensive sequence comparisons among 56 species, including representatives of all major clades of the family and related families. We determine the boundaries of the 22-kb (now known as 22.8 kb) inversion that defines a major split within the Asteraceae, and in the process, we characterize the second and a new, smaller 3.3-kb inversion that occurs at one end of the larger inversion. One end point of the smaller inversion is upstream of the trnE-UUC gene, and the other end point is located between the trnC-GCA and rpoB genes. Although a diverse sampling of Asteraceae experienced substantial length variation and base substitution during the long evolutionary history subsequent to the inversion events, the precise locations of the inversion end points are identified using comparative sequence alignments in the inversion regions. The phylogenetic distribution of two inversions is identical among the members of Asteraceae, suggesting that the inversion events likely occurred simultaneously or within a short time period shortly after the origin of the family. Estimates of divergence times based on ndhF and rbcL sequences suggest that two inversions originated during the late Eocene (38–42 MYA). The divergence time estimates also suggest that the Asteraceae originated in the mid Eocene (42–47 MYA). 1784 Kim et al. FIG. 1.—Physical map showing the cloned fragments and the gene orders of the Lactuca sativa chloroplast genome (adapted from Jansen and Palmer 1987a). Regions containing the four fragments (7.7, 7.2, 7.1, and 6.7 kb) were sequenced to characterize the two inversions. Inverted repeat (IR), small single copy (SSC), and large single copy (LSC) regions are shown below the cloned fragments. Arrows indicate the direction of transcription of chloroplast genes. (version 1.21, Lowe and Eddy 1997) and MFOLD (version 3.0, Zuker 2003). Repeated sequences were identified using REPuter (Kurtz et al. 2001). Fifty-six species, representing all major clades of Asteraceae and seven related families, were selected for comparative sequencing of inversion end points. DNA was isolated from 1–3 g of leaf tissue using the cetyltrimethyl ammonium bromide (CTAB) method (J. J. Doyle and J. L. Doyle 1987), followed by purification using cesium chloride-et (...truncated)