chromosome differentiation and genome organization in

Chromosome differentiation and genome organization in carnivorous plant family Droseraceae

Junichi Shirakawa1, Yoshikazu Hoshi2,4 and Katsuhiko Kondo3

1Graduate School of Bioscience, Tokai University, Kawayou,Minamiaso-mura, Aso-gun, Kumamoto 869-1404, Japan;

2Department of Plant Science, School of Agriculture, Tokai University,Kawayou, Minamiaso-mura, Aso-gun, Kumamoto 869-1404, Japan;

3Laboratory of Plant Genetics and Breeding Science, Department of Agriculture, Faculty of Agriculture, Tokyo University of Agriculture, Funako 1737, Atsugi City, Kanagawa 243-0034, Japan

4Author for correspondence: ([email protected])Received October 5, 2011; accepted November 28, 2011

ABSTRACT: A carnivorous plant lineage including the Droseraceae and its closely related family Drosophyllaceae shows wide range of chromosome size or genome size, whereas all other carnivorous families possess rather small in the genomes sizes. This study gives an overview of the genome size diversity in carnivorous plant families, and evolutional trend of chromosome differentiation especially in the Doroseraceae.

KEYWORDS: Chromosome size, Doroseraceae, FISH, Genome

Chromosome Botany (2011) 6: 111-119© Copyright 2011 by the International Society of Chromosome Botany

The diversities of the chromosome and genome sizes in angiosperms have generated considerable interest over the years (Bennett and Leitch 2005; Leitch et al. 2007; Leitch and Bennett 2007). Nevertheless, little work has focused specifi cally on carnivorous plant families. There are still a lack of C-value data and an uneven karyomorphological knowledge in the carnivorous plant families. For example, the fi rst C-value record for carnivorous plant was originally given by Rothfels and Heimburger (1968), nearly 20 years after the fi rst plant genome size estimation (Ogur et al. 1951). In contrast, the chromosome numbers of the plants, especially Drosera species, were known in the early 1900s (Rosenberg 1903, 1904, 1909). Up to the present date, the Plant DNA C-values Database and the Index to Plant Chromosome Number, which are most available database sites, have contained only 7.2% and 22.2% of the informative data in all carnivorous plant species, respectively. The carnivorous plant family Droseraceae historically includes four genera, Aldrovanda, Dionaea, Drosera and Drosophyllum (Diels 1906; Cronquist 1981), although some morphological studies (Takahashi and Sohma 1982; Juniper et al. 1989; Conran et al. 1997) and molecular phylogenetic analyses (Albert et al. 1992; Williams et al. 1994; Rivadavia et al. 2003) suggest that Drosophyllum does not belong to the Droseraceae. Three genera are monotypic plants, and only genus Drosera comprises more than 100 species distributed mainly in the Southern Hemisphere, with some in the Northern Hemisphere (Juniper et al. 1989; Lowrie 1998). First, thus, C-values and chromosome sizes for 48 and 134 out of 582 species in 12 families were used to provide an overview of the genome diversity and chromosome differentiation in these highly specialized plants, especially in the Doroseraceae. The present work also adds new C-value data obtained by

fl ow cytometry and microscopic chromosome data to our limited knowledge of genome size in carnivorous plants.　 Concerning high genome diversity, special chromo-somal feature called ‘diffuse centromeric type’ found in Drosera should be noted. The chromosomes of most eukaryotes have ‘localized centromere’ which presents as a primary constriction. Whereas, in some plant genera such as Luzula (Juncaceae) (Castro et al. 1949) and Eleocharis (Cyperaceae) (Håkansson 1958), ‘non-localized centromere’ or ‘diffuse centromere’ are known. It does not show any constriction or localized centromere position on chromosome. In Drosera, distinct primary constriction, localized centromere or clear chromosomal gap between sister chromatid has not been observed in the past, supporting the diffuse centromere hypothesis (Kondo 1976; Kondo et al. 1976; Kondo and Lavarack 1984; Kondo and Segawa 1988). Since the diffused type possesses centromere function dispersed along the whole chromosome length, in theory all fragments of this type of chromosomes are stably transmitted after cell division. Due to existing intraspecifi c aneuploid cytotypes with several fragmented chromosomes, chromosome differentiation event such as section Bryastrum and section Lasiocephala of Drosera is able to explain using diffuse centromeric hypothesis, but not section Drosera due to showing strict about rules of poliploidization. A well demonstration of the diffuse centromeric chromosome has done to test the ability of regular cell division and accurate segregation of each chromosome fragment, induced by Gamma- or X-ray irradiations in Luzura (Castro et al. 1949). In Drosera, Gamma-radiated plants of Austrarian anuploidal species have also shown evidence that typical segregations of fragments and minute chromosomes at mitotic anaphase stages are observed in mitotic cell division (Sheikh et al. 1995; Furuita and

112 SHIRAKAWA ET AL.

Kondo 1999), confi rming the validity of the hypothesis proposed by earlier workers (Kondo et al. 1976; Kondo and Lavarack 1984). However, there is no experiment using polyploid materials in Drosera species. To discuss the chromosome differentiation, this work also gives the data of Gamma-irradiated chromosome behaviors both of D. petioralis as Austrarian aneuploid group and D. rothundifolia, representative species in the Northern Hemisphere, as polyploid group.

MATERIALS AND METHODSPlant materials　Plant materials in most Drosera species used in our experimental study are cultivated in greenhouse and some strains are cultured on hormone-free 1/2 Murashige and Skoog basal medium (Murashige and Skoog 1962) supplemented with 0.35% gellan gum and 3% sucrose for in vitro culture.

Flow cytometry　Performance validation of the EPICS XL ADC fl ow cytometer was done using fl uorescent bead mixtures provided by the manufacturer, and according to their instructions (Beckman Coulter). Analysis was based on light-scatter and fl uorescence signals produced from 20 mW laser illumination at 488 nm. Signals corresponding to forward angle- and 90o-side scatter (FALS, SS) and fl uorescence were accumulated, the fl uorescence signals (pulse area measurements) being screened by the following fi lter confi gurations: (a) FL-2: a 585/40 nm band-pass fi lter, and (b) FL-3: a 670 nm longpass fi lter. Threshold levels were empirically set (10,000 for FALS, with a second threshold of 1,000 for FL-2) to eliminate from detection the large amounts of irrelevant debris that are found in plant homogenates. Templates for uni- and bi-parametric frequency distributions were established, and on identifi cation of the region corresponding to nuclei, data was collected to a total count of 5,000-10,000 nuclei. The fl ow cytometer was routinely operated at the Slow Flow Rate setting (14 lL sample/minute), and data acquisition for a single sample typically occupied 3-5 min.

Chromosome slide preparation　After root tips were pretreated with 0.2 mM 8-hydroxyquinoline for 2 h at 18 oC, they were fi xed in 70% ethanol for 1 h on ice, washed with distilled water for 60 min, and then macerated in an enzymatic mixture containing 4% Cellulase Onozuka RS (Yakult Pharmaceutical Industry Co., Ltd., Tokyo, Japan) and 2% Pectolyase Y-23 (Seishin Pharmaceutical Co., Tokyo, Japan) for 1 h at 37oC. After washing with distilled water for 1 h, root tips were placed onto glass slide, and spread with ethanol-acetic acid (3:1). The preparations were air-dried for 24 h at room temperature.

Fluorescent staining with CMA and DAPI　Chromosome preparations were stained with 25 μg/ml chromomycin A3 (CMA) (Sigma-Aldrich Inc., MO, USA) in McIlvaine’s

buffer (pH 7.0) containing 5 mM MgSO4 and 50% glycerol. These chromosome preparations stained with CMA were observed with a BV fi lter. Then, the slides were used for sequential 4’,6-diamidino-2-phenylindole (DAPI) (Nacalai Tesque, Inc., Kyoto, Japan) staining. The slides were destained in 45% acetic acid for 30 min, dehydrated in a series of ethanol, and air-dried for 30 min. They were stained with 1 µg/ml DAPI in McIlvaine’s buffer containing 50% glycerol. The chromosomes stained with DAPI were observed with a U fi lter. Chromosome sizes (lengths) on digitally-recorded chromosome images were determined using ImageJ 1.33u (National Institutes of Health, MD, USA).

Gamma radiation treatment　Plants of Drosera petiolaris and D. rotundifolia in vitro were exposed to Gamma radiation in the Facility of Faculty of Engineering, Hiroshima University. Doses were determined by time and distance from the source around a circular disc in the ‘Gamma shine’ (Co unit). Soon after exposed to Gamma radiation, the fl asks were returned to the culture room of the Laboratory for continuous in vitro culture.

RESULTS AND DISCUSSIONGenome diversities of the Droseraceae and other carnivorous plant families　Table 1 shows all genome size information in carnivorous plants with our new data. Even including our new experimental data, 1 C-values in Drosera are known only for nine species ranged from 0.30 pg in D. capensis to 0.95 pg in D. intermedia (if the related genera are included, in this case, max value is Drosophyllum lusitanicum with 15 pg) (Table 1). All information of the chromosome numbers of 66 out of 142 species in the Droseraceae, including Drosophyllum show that each carnivorous family posses rather small genome size less than 1 pg, but not the Droseraceae. In case of including Drosophyllum, they are quite variable angiosperm lineage with values ranging 50 fold from 1C, and 20 fold from average chromosome size. Given this, it suggests that genome evolution in the Droseraceae and its related lineage is dynamic.

Superimposing chromosome information and the genome size data onto the molecular phylogenic tree of the Droseraceae　The intrageneric classifi cation of Drosera had been problematic for a long time (Rivadavia et al. 2003), in addition to confused intergeneric relationships among the Drosera and two other monotypic genera in the Droseraceae with snap traps, Aldrovanda and Dionaea. With respect to these phylogenic problems, molecular analysis of large data sets to compare DNA sequencing has become a widespread tool for resolving taxonomic problems and for phylogenic reconstruction on all levels. To date, covering Aldrovanda, Dionaea and all Drosera sections except one, molecular phylogenic analyses using nuclear and chloroplast DNA sequences have performed

113 CHROMOSOME DIFFERENTIATION IN DROSERACEAE

Family Genusa SpeciesbChromosome number Experimental

1C-value (pg)Genome size

(Mbp)c2n n

Bromeliaceae Brocchinia - - - -Catopsis - - - -

Byblidaceae Byblis B. linifl ora 32 8 0.89 872Cephalotaceae Cephalotus C. follicularis 20 10 0.64 627Dioncophyllaceae Triphyophyllum T. peltatum - - - -Drosophyllaceae Drosophyllum D. lusitanicum 12 - 15.00 14700Droseraceae Aldrovanda A. vesiculosa 48 - 0.52* 510

Dionaea D. muscipula 32 - 0.72* 706Drosera D. arcturi 58 - 0.55* 539

D. binata 32 - 0.63 617D. capensis 40 10 0.30 294D. intermedia 20 10 0.95 931D. linearis 20 10 0.93 911D. rotundifolia 20 10 0.88 862

20 1.37 1338D. regia 34 - 0.56* 549D. spatulata 40 - 0.55* 539

Eriocaulaceae Paepalanthus - - - -Lentibulariaceae Genlisea G. aurea ca. 52 - 0.06 59

G. hispidula - - 1.54 1509G. lobata 16 - 1.31 1284G. margaratae ca. 40 - 0.06 59G. uncinata - - 1.02 1000G. violacea - - 1.03 1009

Pinguicula P. agnata 22 - 0.76 745P. cyclosecta 22 - 0.53 519P. ehlersiae - - 0.50 490P. emarginata 22 - 0.81 794P. esseriana 32 - 0.85 833P. gracilia - - 0.61 598P. gypsicola 22 - 0.56 549P. heterophylla 22 - 0.53 519P. macrophylla 22 - 0.71 696P. primulifl ora - - 0.68 666

Utricularia U. australis 18, 19, 20, 22 - 0.18 176U. blanchetii - - 0.14 137U. gibba - - 0.09 88U. humboldtii - - 0.22 216U. livida - - 0.26 255U. microcalyx - - 0.18 176U. parthenopipes - - 0.14 137U. praelonga - - 0.16 157U. prehensillis - - 0.41 402U. pubescens - - 0.22 216U. quelchii - - 0.16 157U. reniformis - - 0.34 333U. sandersonii - - 0.24 235U. subulata - - 0.25 245

Martyniaceae Ibicella - - - -Nepenthaceae Nepenthes N. pervillei 80 - 0.28 274Roridulaceae Roridula R. gorgonias 12 6 0.19 186Sarraceniaceae Darlingtonia D. californica - - - -

Heliamphora - - - -Sarracenia S. fl ava 26 - 4.35 4263

Table 1. Chromosome numbers and genome sizes of carnivorous plant families

*: Present data. All other 1C-values were from Angiosperm DNA C-values database (mostly from Rothfels and Heimburger 1968), except for higher value in D. rotundifolia (Greilhuber 2008).a: All carnivorous plant genera were listed.b: Only species with 1C-value data were listed.c: 1 pg = 980 Mbp.


(Rivadavia et al. 2003). These analyses reveale that 1) all Drosera species form a clade sister to a clade including Dionaea and Aldrovanda, 2) fi ve of 11 sections in Drosera are polyphyletic, and 3) most species native to Australia are clustered basally. These outcomes indicate that Australian species expanded their distribution to South America and then to Africa. And then, expansion of distribution to the Northern Hemisphere from the Southern Hemisphere occurred in a few different lineages.　 Moreover, superimposing chromosome data with genome size information onto the Droseraceae phylogenic context shows new insight of genome size diversity and chromosome evolution of this family (Fig. 1). This fi gure shows that multiple episodes of most aneuploidy occurred in a basal clade that includes mainly Australian species, while multiple episodes of polyploidy occurred in a derivative clade that includes mainly South America, Africa and Northern Hemisphere species. 　 Although the relationship between genome size and

total chromosome length in a karyotype are not precise (Bennett and Rees 1967, 1969), there are often a correlation between these two parameters, enabling chromosome data to be used as rough proxies for genome size. Thus, putative C-values of species in the Droseraceae were proposed to calculate using previous information of total chromosome length. The putative values are given to 40 species to clarify relationships between genome size and chromosome size in the Droseraceae (Fig. 2). In spite of D. capensis with experimental minimum C-values so far, the putative data indicates that D. burmannii seems to have the smallest genome size (putative 1C value is 0.21 pg). A recent critical review for genome size estimation pointed out that secondary metabolites make fl ow cytometric determinations as well as Feulgen measurements problematic, especially in the Droseraceae (Greilhuber 2008). Greilhuber (2008) was determined a 2C-value of 2.73 pg in D. rotundifolia, which is higher than a value of 1.76 pg published by Rothfels and

Fig. 1. Chromosome size (average of individual length) and genome size (C-value) data for the Droseraceae onto a morecular phylogenic tree based on Rivadavia et al. (2003). Experimental 1C-value so far was put as genome size in this fi gure, but not the putative value calculated by total chromosome length.


Heimburger (1968). This work also noted using an improved Feulgen method, that C-values in Droseraceae published by Rothfels and Heimburger (1968) are likely underestimates due to tannin error. Although out genome size study is premature for precise estimation in Drosera species, comparison of somatic chromosome number with genome size DNA amounts provides some interesting clue as to understanding of genome size diversity and chromosome changes in the Droseraceae (Fig. 2). The comparative result indicates that 1) aneuploid group such as section Bryastrum and section Lasiocephala (infrageneric classifi cation is based on Seine and Barthlott 1994) shows narrow range (less than 5-fold) of larger genome size (Fig. 2b), even they have wide range with a series of different chromosome numbers of 2n=6-28, 2) polyploid group with basic chromosome number of x=10 such as section Drosera shows wide range (more than 10-fold) of smaller genome size. Additionally, the polyploid chromosome numbers among closely related species are positively correlated with DNA amounts of putative C-values. These suggests that chromosome fi ssion or

chromosome fusion without any changing of DNA amount are signifi cant role for genome differentiation in aneuploid series, while chromosome doubling with an integral multiple increase of genome size is primary event for speciation of polyploidal series. Given this, genome size evolution of Drosera is quite dynamic. Such intragenomic diversity is rather rare in other carnivorous plant groups. For example, no difference of chromosome number is found in genus Nepenthes (all species so far researched are 2n=80). Especially, the diversity both with the cases of aneuploid and polyploid chromosome differentiation like in Drosera is quite restricted to plant genus, even in a level of the family contained a large number of species.

Drosera chromosomes with diffuse centromeric nature　In our experiments, axenic-cultured Drosera petiolaris with 2n=14 and D. rotundifolia with 2n=20 were exposed to various doses of Gamma radiation and occurred fragmentation at dose more than 10 Gy radiation of Gamma-ray (Figs. 3 and 4). Whereas the plant materials in vitro exposed to above 1000 Gy were completely died

Fig. 2. Total chromosome length (a), putative C-value (b) and putative haploidal C-value (c) of 41 species and one natural hybrid in the Droseraceae. Drosophyllum lusitanicum (2n=12) was excluded from this fi gure due to having huge values (Total chromosome length, C-value and putative haploidal C-value in Drosophyllum lusitanicum were 150.9 µm, 15.0 pg and 9.4pg, respectively).


120 days after the exposure. In exposed to 50 Gy, more than 90% of the metaphase cells both of D. petiolaris and D. rotundifolia had chromosome abbreviations (Fig. 3a). They showed chromosome numbers more than those of

the mother plant. Simple breakages occurred frequently in most of the metaphase cells exposed to 50 Gy produced chromosome fragments with various lengths. Chromosome fragments with various sizes included minute

Fig. 3. Gamma irradiated experience of Drosera petiolaris and D. rotundifolia. More than 90% of mitotic cells in both species exposed to 50 Gy keep regular division with chromosome abbreviations with fragment chromosomes (vertical bars represent the standard error) (a), while chromosome fragments per cell after exposed to 50 Gy vary 6.43 in the D. petiolaris chromosome complement and 4.72 in the D. rotundifolia chromosome complement (b). The boxplot represents the mean (central solid line), median (central dotted line), 25th and 75th percentage (box), and out liers.

Fig. 4. Mitotic chromosome complements in Drosera petiolaris (a, c-h, and k-o) and D. rotundifolia (b, i and j) before and after Gamma irradiation. a and b. Metaphase chromosomes in the control. c and i. Metaphase chromosomes after exposure to 10 Gy. d-g and j. Metaphase chromosomes after exposure to 50 Gy. h. Metaphase chromosomes after exposure to 100 Gy. k-n Early-anaphase to late-anaphase chromosomes after exposureto 50 Gy. o. Interphase nucleus and two minute nuclei after exposure to 50 Gy. Bar = 5 µm.


chromosomes less than 0.5 µm long were found in mitotic metaphase cells in D. petiolaris and D. rotundifolia more than 300 days after irradiated. Moreover, sister chromatids less than 0.5 µm long were observed to separate parallel normally in early anaphase (Fig. 4m). Regular disjunction and proper segregation of the fragmented chromosomes were observed at early anaphase. A number of fragment chromosomes less than 0.5 µm long were found in every metaphase cell. On the other hand, long chromosomes made by fusion of two or more chromosomes were also found in some metaphase cells radiated. After exposed to 50 Gy, maximum chromosome numbers of D. petiolaris and D. rotundifolia were 25 and 30, respectively. Moreover average number of fragments per cell was 6.43 in the D. petiolaris chromosome complement and 4.72 in the D. rotundifolia chromosome complement (Fig. 3b).　 The localized-centromeric chromosomes always display lagging chromosomes and/or chromosome bridges at mitotic anaphase and micronuclei at mitotic telophase after irradiation (Rieger et al. 1976), but not these Drosera species. Moreover, the behavior and appearance of the fragmented chromosomes are quite in agreement with some of the features of holocentric and/or diffused-centromeric chromosomes reported previously (Sheikh et al. 1995; Murakami and Imai 1974; Tanaka and Tanaka 1977). The present data, thus, indicates that the separation of the fragment chromosomes at mitotic anaphase and telophase are quite normal, strongly suggesting the diffuse centromeric chromosome both of species. 　 In spite of possessing diffuse centromeric features with numerous fragments both in aneuploid group and poliploid group, different features of gamma-radiation between two species are shown in frequencies of chromosome aberrations with fragment chromosomes. Speciation accompanied by orderly and stable polyploidization in Drosera seen in the Northern Hemisphere can be an unexpected phenomenon. This may be due to difference of centromeric behavior function between two groups. A meiosis study shows the bivalent chromosome shapes are diffused type in aneuploidal species, but not polyploidal species (Hoshi and Kondo 1998b). Polyploid species provide typical shape of localized centromeric bivalent chromosomes in the meiosis, instead of diffused-centromeric nature seen during the mitosis. Thus, centromere differentiation as meiotic separating mechanism seems to be also occur during speciation and evolution in phylogenetically derivative species with basic chromosome number of x=10.

Evolutional trend of chromosome-size and genome-size changing　According to cytogenetic studies, localized centromere and diffused centromere have been seen in the Droseraceae, although centromeric type of chromosome is generally conservative in the plant genus level. In the Doroseracese, Dionaea muscipula and Drosophyllum

lusitanicum have the localized-centromeric chromosome, while Aldrovanda vesiculosa and all Drosera species show the chromosomes with no primary constriction. Moreover, current chromosome study shows that a centromere-like primary constriction on somatic chromosomes in phylogenically basal Drosera species, D. regia (Shirakawa et al. 2011). Figure 5 illustrated the centromere types onto a framework of phylogenic relationships in the Droseraceae. Thus, the founding of primary constriction in the genus Drosera suggests that centromere differentiation may be independently occur not only in the Droseraceae, but also in genus Drosera. 　 Terminologically, it is important to discriminate between nucleic DNA amount (n or 2n) and DNA amount of basic chromosome number (x). Greilhuber et al. (2005, 2006) mentions that 1C is the DNA content of the unreplicated holoploid complement with n chromosomes, while 1Cx is the DNA content of the unreplicated monoploid set of x chromosomes (x being the basic chromosome number of a polyploid series). Because higher polyploidy is seen in Drosera, putative 1Cx values are also estimated in this paper (Fig. 2c). Genome size of basic chromosome number (1Cx), which is obtained by compare basic chromosome number and putative C-value, is particularly useful for provide farther insight of Drosera chromosome differentiation, especially in poliploidal groups. In section Drosera with basic chromosome number of x=10, present result shows that the genome size data clearly distinguish one group from another (Fig. 2c). As expected, regardless of level of ployploidy, the species with genome sets comprised of all small size chromosomes (S-type) show smaller DNA amount of basic set of chromosomes, while the species with genome sets comprised of all middle size chromosomes (M-type) show larger DNA amount of basic set of chromosomes (Fig. 2c). Moreover, notheworthy point of view is in the direction of chromosome evolution. Combined with the

Fig. 5. A phylogenic relationships of the Droseraceae with its several types of chromosomes stained with Chromo-mycin A3 (CMA) and 4´, 6-diamidino-2´-phenylindole (DAPI).


molecular tree (Rivadavia et al. 2003) and many cytological papers with Drosera chromosome size, evolutional trend of the large increase of chromosome size and genome size emerges in the several Northern Hemisphere species possessed M-type chromosomes (Hoshi and Kondo 1998a; Hoshi and Kondo 1998b; Hoshi et al. 2008, 2010). Evolutionally chromosome-size increase is rather common event in angiosperm, although the evolutional size decrease with DNA amount is rarely seen in some plants families such as Malvaceae (Wendel et al. 2002), Brassicaceae (Johnston et al. 2005) and Poaceae (Price et al. 2005). The mechanisms of the DNA loss are, however, not clearly understood because the number of examples are still low. 　 The size changing is considered to be greatly affected by the amounts of repetitive sequences and ploidy levels in plant species (Bennetzen 2000; Grover and Wendel 2010). Although the difference of ploidy levels gives just a vast change with doubling of genome sets, the difference in the number of repetitive DNA sequences makes increase or decrease with in haploidal genome set of basic chromosome number. Thus, an increase-decrease event of the repetitive sequences, which is related to transposable elements, directly contributes to unreplicated nomoploidy chromosome set in plant genome. Additionally, transposable elements, especially retrotransposons, cause repetitive DNA dispersing uniformly over all chromosomes of the genome (Kamm et al. 1996; Galasso et al. 1997; Heslop-Harrison et al. 1997; Miller et al. 1998; Vicient et al. 1999; Hanson et al. 2003). Indeed, the retrotransposons for size increasing occupied more than 40% and 90% of the genome in Vicia (Pearce et al. 1996) and Triticum (Flavell 1986), respectivelly. Transposons make the amplifi cation and activation in the genome, and therefore can be correlated with biodiversity, genetic variation and eventually speciation (Galasso et al. 1997; Flavell et al. 1997; Heslop-Harrison and Schwarzacher 2011). 　 With a number of recent reviews as genome size changing among a variety of species, Grover and Wendel (2010) presents interesting ideas of genome change factors with environmental effects or ecological infl uences such as population size, population bottleneck and drift, as well as epigenetic factors with DNA methylation and heterochromatinization of chromosome segment. From the perspective of environmental effect, Hanson et al. (2001) already pointed out a mineral-poor environment regarding to the genome size reduction in carnivorous plants. It may impose pressure to reduce redundant phosphorus-rich nucleic acid such as non-essential repeated DNA sequences, and hence tend to minimize genome sizes. Distinctive differences with genome size are seen between the Drosera and Drosophullum. Hanson et al. (2001) also noted that Drosophullum was more able than Drosera to acquire both scarce water and phosphorus from the soil, supporting the hypothesis of genome size reduction in carnivorous plants.

　 Concerning future genome works with the next generated technologies, species of the Droseraceae are useful genetic resources to introduce the evidence and new insight for genome organization and chromosome evolution not only in carnivorous plants, but in angiosperms. Further experimental study for the genome size estimation and molecular cytogenetic characterization will be necessary.

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