C2001 The Japan Mendel Society Cytologia 66: 307-311, 2001 Flow Cytometric Determination of Genome Size in the Taxodiaceae, Cupressaceae sensu stricto and Sciadopityaceae Masahiro Hizume1,*, Teiji Kondo2, Fukashi Shibata1 and Ryoko Ishizuka3 1Faculty of Education, Ehime University, Matsuyama 790-8577, Japan 2Forest Tree Breeding Center, Juo, Ibaraki 319-1301, Japan 3Tsukuba Technical Center, Ikeda Science Co., Tsukuba 305-0062, Japan Accepted August 3, 2001 Summary The genome sizes of 13 species of Taxodiaceae, 19 species of Cupressaceae s.s. and Sciadopitys verticillata were determined by flow cytometry of isolated nuclei stained with propidium iodide, using Hordeum vulgare nuclei as an internal standard. In Taxodiaceae, the genomes of Cun- ninghamia lanceolata (28.34 pg/2C) and Taiwania species (25.78, 26.80 pg/2C) were larger than those of other genera/species, which ranged from 19.85 to 22.87 pg/2C. In Cupressaceae s.s., genome size ranged from 20.03 to 27.93 pg/2C among 16 species. The Calocedrus species and Thu- jopsis had a larger genome than most other species. Sciadopitys verticillata had a large genome of 41.60 pg/2C. After comparing the diversity in genome size with previously reported cladograms con- structed using nucleotide sequence data, the tendency of changes in genome size with phylogenetic differentiation is discussed. Key words Cupressaceae, Flow cytometry, Genome size, Taxodiaceae, Sciadopitys. All genera of Taxodiaceae, except for Athrotaxis, are distributed in north-temperate to subtrop- ical regions of the North Hemisphere and most genera are growing in mountainous areas around the Pacific Ocean. Glyptostrobus, Metasequoia, Sequoia, and Sequoiadendron are monotypic genera, and the other genera include only a few species. All species of Taxodiaceae seem to be endemic. This family has been extensively studied cytologically (Schlarbaum and Tsuchiya 1984, Hizume et al. 1988, Hizume 1989). The basic chromosome number of this family is x=11. While nearly all species are diploid, Sequoia sempervirens is a hexaploid species, and triploid trees occur sponta- neously in Cryptomeria japonica, Glyptostrobus pensilis, and Taiwania cnyptomerioides. Genome size is important in the analysis of genome and phylogeny. In Taxodiaceae, genome size has been reported in 9 species by estimation from the interphase chromosome volume (Price et al. 1973), in 3 species by microphotodensitometry (Ohri and Khoshoo 1986) and in 1 species by flow cytometry (Sasaki et al. 1997). Direct measurement of genome size in many species of Taxodiaceae by cy- tophotometry or flow cytometry under the same experimental conditions is desirable. A recent taxo- nomic treatment of Taxodiaceae and Cupressaceae s.s. reported the results of several methods (Gadek et al. 2000). Recent molecular phylogenetic analyses indicate that Cupressaceae s.l. in- cludes Taxodiaceae and Cupressaceae s.s. (Brunsfeld et al. 1994, Tsumura et al. 1995, Gadek et al. 2000, Kusumi et al. 2000). The genome sizes of Cupressaceae s.s. have been reported for several species (Murray 1998). This study is aimed at determining the genome sizes of all genera in Taxodiaceae, and in re- vealing the diversity of genome size in this family and related families in order to supply informa- tion for genome and phylogenetic studies. The genome sizes of 13 species of Taxodiaceae, 19 species of Cupressaceae s.s. and Sciadopitys verticillata were measured by flow cytometry after * Corresponding author, e-mail: [email protected] 308 Masahiro Hizume et al. Cytologia 66 propidium iodide staining. Materials and methods Genome size was measured in 13 species of 9 genera in Taxodiaceae, 19 species of 10 genera in Cupressaceae s.s. and Sciadopitys verticillata. The species studied are listed in Table 1. Plants of Taxodiaceae genera, except for Cryptomeria and of Sciadopitys, Fokienia, Microbiota and Caloce- drus were cultivated in the experimental garden at the Faculty of Education, Ehime University, Mat- suyama, Japan. Other species of Cupressaceae s.s. and Cryptomeria species were cultivated at the Forest Tree Breeding Center, Juo, Ibaraki, Japan. Seeds of Sequoiadendron giganteum came from California, USA, and those of Athrotaxis species were from Tasmania, Australia. Seedlings of Hordeum vulgare cv. Minorimugi were used as a standard sample (11.12 pg/2C, Bennett et al. 1982). Small pieces of young leaves of the target species, and of H. vulgare as an internal standard, were chopped with a razor blade in 400 ƒÊl of extraction buffer (Cystain PI absolute P, Partec) and filtrated through a 50 ƒÊm nylon mesh filter (Cell Trics). Then 1.6 ml of staining buffer containing 62.5 ƒÊg/ml propidium iodide and 0.02ƒÊg/ml RNase A were added, and the suspension was left at room temperature for 60 min. The suspension was analyzed with a flow cytometer (CCA, Partec). Results and discussion Leaf nuclei of 13 species of 9 genera in Taxodiaceae, 19 species of 10 genera in Cupressaceae s.s. and Sciadopitys verticillata in Sciadopityaceae were analyzed for their DNA content by flow cy- tometry. In order to avoid the influence of differences in the base Composition of the genome among species and of the presence of RNA on the determination of genome size, propidium iodide staining and RNA digestion were performed at the same time. Nuclei of sample species were mixed with those of Hordeum vulgare as an internal standard and measured simultaneously. No polyploidy peak appeared in any experiment, which indicates that endopolyploidy does not occur in the leaf tissues of the conifer species examined. The genome size was measured twice in one or more plants of each species and the mean values are summarized in Table 1. The genome size of Glyptostrobus, Taxodium and Sequoiadendron was about 20 pg/2C. A hexaploid species, Sequoia sempervirens, gave 64.27 pg/2C and the diploid level estimated was 21.42 pg. The genome size of Metasequoia, Cryptomeria and Athrotaxis was about 22 pg/2C. The diploid genome sizes of Cunninghamia lanceolata (28.34 pg/2C) and Taiwania species (25.78 and 26.80 pg/2C) were the largest among Taxodiaceae. The genome sizes of 3 species have been determined by microphotodensitometry after Feulgen staining (Ohri and Khoshoo 1986), and that of one species by flow cytometry (Sasaki et al. 1997). These values are also given in Table 1. Price et al. (1973) estimated genome sizes from nuclear volume in 9 species, but this indirect estimation is not accepted as a standard method. Sasa- ki et al. (1997) used DAPI-staining in their flow cytometric analysis. Since DAPI stains adenine and thymine specifically, this stain should be used to analyze ploidy levels among individuals or among tissues in the same species, but not to determine the genome size in species with different base compositions. Our flow cytometric values are close to values obtained by microcytophotometric de- termination, except for those for Metasequoia. Since our flow cytometric experiments were per- formed under the same conditions and using a mixed internal standard, the genome size determined in our study is considered closest to the real value. In a karyotype analysis, Cunninghamia and Taiwania ctyptomerioides were shown to have a larger total chromosome length than other species in Taxodiaceae (Hizume 1989). Although a chro- mosomal parameter such as chromosome length varies somewhat due to contraction of chromo- somes during pretreatment and artificial stretching during stages of the preparation process, such as squashing, in general, chromosome lengths match the genome sizes measured here. 2001 Genome Size in Taxodiaceae, Cupressaceae and Sciadopityaceae 309 Table 1. Measurements of genome size in Taxodiaceae, Cupressaceae s.s. and Sciadopityaceae All molecular systematic studies indicate that Taxodiaceae and Cupressaceae s.s. are a mono- phyletic group, Cunninghamia is the basal lineage of Taxodiaceae, Cryptomeria, Glyptostrobus and Taxodium form a group, and Metasequoia, Sequoia and Sequoiadendron form a group (Brunsfeld et al. 1994, Tsumura et al. 1995, Gadek et al. 2000, Kusumi et al. 2000). The most parsimonious tree reported by Kusumi et al. (2000) indicates that after segregation of Cunninghamia, Taiwania segre- gated, followed by Athrotaxis. Then the Metasequoia/Sequoia/Sequoiadendron group segregated and the CryptomerialGlyptostrobus/Taxodium group and Cupressaceae s.s. diverged. Generic segre- gation was compared with variation in genome size in Taxodiaceae, as follows: Cunninghamia lanceolata has the largest diploid genome in Taxodiaceae and the genome of Taiwania is 25.78 and 26.80 pg/2C. Other genera or generic groups, Athrotaxis, MetasequoialSequoialSequoiadendron and CryptomerialGlyptostrobus/ Taxodium, have small genomes (19.85-21.87 pg/2C) at the diploid 310 Masahiro Hizume et al. Cytologia 66 level. This phenomenon strongly suggests that the genome size decreases according to generic seg- regation in Taxodiaceae. In the Metasequoia/Sequoia/Sequoiadendron group, Sequoiadendron seg- regated from Metasequoia and in the Cryptomeria/Glyptostrobus/Taxodium group, Taxodium and Glyptostrobus segregated from Cryptomeria. These genera seem to be the most recently differenti- ated among Taxodiaceae genera. The deciduous nature of Taxodiumand Glyptostrobus is indicative of their close phylogenetic relationship, but that of Metasequoia seems to have evolved indepen- dently, as an adaptation to a cold, dry climate. With regard to karyotype evolution, these results also suggest that the somewhat heterogeneous karyotype in