Chromosome Botany (2007) 2: 39-43 © Copyright 2007 by the International Society of Chromosome Botany

A comparative study of karyotypes in two species of (Byblidaceae)

Yoshikazu Hoshi1, Kenji Fukushima and Katsuya Nagano

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

1Author for correspondence ([email protected]) Received December 5, 2006; accepted January 26, 2007

ABSTRACT. Byblis filifolia and B. liniflora had the diploidal and the tetraploidal chromosome numbers of 2n=16 and 32, respectively. In condensation behavior from prophase to metaphase, most chromosomes of both species had early condensing segments at the proximal regions. At prometaphase and early-metaphase, all chromosomes except for some small sized chromosomes had decondense segments at the distal regions in both arms. At mid-metaphase, well-spread chromosomes were quite rare to obtain, because all chromosomes at mid-metaphase stage were quite sticky nature, but not earlier stages of metaphase and prophase. The metaphase chromosomes in both species showed a gradual decrease in size from the largest to the smallest. In B. filifolia, total chromosome length at early-metaphase was 40.5 µm, while total chromosome length at mid-metaphase was 23.3 µm. In contrast, B. liniflora showed total chromosome length of 106.1 µm at early-metaphase, and 54.0 µm at mid-metaphase. To compare to the same stage of metaphase, the total length in B. liniflora was nearly twice as long as that of B. filifolia. Two sat- chromosomes were observed in both species.

KEYWORDS: Byblidaceae, Byblis, Chromosome, Karyotype

Byblis is the member of the monogeneric, carnivorous mosome numbers (Conran et al. 2002). The earliest chro- family Byblicaceae mainly distributed in Australia. The mosome count of 2n=18 was done in perennial species genus has long been recognized to consist of only two by Kress (1970). Then, different counts of the chromo- species by several workers (e.g., Kondo 1973; Peng and some numbers of 2n=24 and 32 were reported in the an- Kenton 1982; Conran et al. 2002). Currently, Byblis has nual B. liniflora (Kondo 1973; Peng and Kenton 1982). been regarded as having several species (Conran et al. The most recent chromosome counts were carried out in 2002; Conran and Christophel 2004). According to the six species of Byblis (Conran et al. 2002). The lowest proposal for amendment of Appendices (Anonymous count of 2n=16 was newly recorded in the annual species 2000) in Convention on the International Trade in Endan- including B. filifolia. It was a half chromosome number gered Species (CITES), the genus taxonomically consist- of B. liniflora with 2n=32. Although the study supported ed of five species. On the other hand, based on current the recent separation of taxa, karyomorphological results publication of Conran and Christophel (2004), this genus showed no clear constriction in all Byblis chromosomes. consists of seven species: two perennial species includ- It implied a difficulty to formulate karyotype in this ge- ing B. gigantea, and five annual species including B. lini- nus. As well as chromosome size determination and flora sensu stricto (Lowrie and Conran 1998; Conran et chromatin condensation pattern analysis, addressing of al. 2002; Conran and Christophel 2004). centromere locality (primary constriction) on chromo- The Byblis liniflora complex has four species, B. some is an essential part of the karyotype construction, aquatica Lowrie et Conran, B. filifolia Planch., B. liniflo- that is one of the most important works in cytogenetic ra Salisb. and B. rorida Lowrie et Conran (Conran et al. studies (Kulak et al. 2002). Despite cytological studies in 2002). Byblis aquatica and B. rorida have been recently the last five decades, only one comparative analysis with recorded, while B. filifolia and B. liniflora have been re- karyotypes (Kondo 1973) was attempted in two Byblis corded in the 1800’s. In spite of morphologically and cy- species. tologically discriminable characters, B. filifolia had been We report here in demonstration of clear centromeric placed in a synonym or a subspecies of B. liniflora for a constrictions on all somatic chromosomes in two species, long time (Conran and Lowrie 1993; Lowrie and Conran B. filifolia and B. liniflora, by employing improved chro- 1998). A fossil Byblidaceae seed from the Eocene was mosome preparation technique. Moreover, karyotypes of found in South Australia, Australia, and was compared these species were constructed for comparative analysis with that of extant Byblis species by scanning electron to justify species relationships, and to speculate chromo- microscope (Conran and Christophel 2004) and was de- somal evolution in the B. liniflora complex. scribed similarity to that of the annual B. filifolia, B. lini- flora and B. rorida, but not B. aquatica. Thus, the specia- MATERIALS AND METHODS tion or relationship among those species in the B. liniflora complex has not yet been well-detected. Seeds of Byblis filifolia and B. liniflora were purchased Up to the present, five species of Byblis showed chro- from Seed Bank of Japanese Carnivorous Plant Society.

39 40 HOSHI ET AL CHROMOSOME STUDY IN BYBLIS 41

Fig. 1. Interphase (a and b) and resting (c and d) nuclei and mitotic chromosomes (e-k) at prophase (e and f), prometaphase (g), early- metaphase (h and i) and mid-metaphase (j and k) in B. filifolia (a, c, e, g, h, j) and B. liniflora (b, d, f, i, k). Bar =5μm.

They were surface-sterilized and sown on sterilized 1/2 Scientific, Inc.) on microscope (Olympus BX51). Karyo- Murashige and Skoog (MS) basal medium (Murashige type formulas at mitotic early- and mid-metaphase were and Skoog 1962) supplemented with 0.2% gellan gum based on the data of measurements of the chromosomes without any growth substance. These germinated taken from the computer-images. Position of the primary from the seeds were subcultured and maintained under in constriction was estimated by arm ratio calculated by vitro condition in Department of Plant Science, School of long arm/short arm. Classification of chromosomes fol- Agriculture, Kyushu Tokai University. lowed Levan et al. (1964). Actively growing root-tips were excised when they were approximately 3 cm long, and pretreated with 0.05 RESULTS % colchicine for 2-6 h at 18oC, before they were im- mersed and fixed in 45% acetic acid for 10 min at o0 C. Interphase nuclei with common feature in the two species They were hydrolyzed in 1 M HCl : 45% acetic acid (2:1) are shown in Fig. 1a and b. They were stained lightly and for 7 sec at 60oC, and transferred onto glass-slide. The had some slightly dark-stained chromocenters. In con- root-cap was removed from root tips, and only a mass of trast, resting nuclei showed many darkly stained chromo- cells in meristematic tissues was stained with 8 µl of 2% centers occasionally aggregating more than 10 and 20 orcein, and immediately squashed. To obtain resting nu- large heteropycnotic blocks in B. filifolia and B. liniflora, clei, root cap tissues were used for squash preparation. respectively (Fig. 1c and d). The blocks varied in size Within 12 to 48 h after making preparation, chromosome from 0.5 to 1.0 µm. images were taken by digital camera (CoolSNAP: Roper Mitotic prometaphase, early-metaphase and mid- 40 HOSHI ET AL CHROMOSOME STUDY IN BYBLIS 41

Fig. 3. Aligments of the chromosome-sets of two species in Byblis at mitotic mid-metaphase. (a). B. filifolia, 2n =16. (b). B. liniflora, 2n =32. Bar =5μm. Fig. 2. Aligments of the chromosome-sets of two species in Byblis at mitotic early-metaphase. (a). B. filifolia, 2n=16. (b). B. liniflora, 2n=32. Bar =5μm.

Table 1. Chromosome numbers of two species of Byblis investigated Chromosome number Species Previous count References Present count (2n) (n) (2n) B. filifolia 16 16 Conran et al. 2002 B. liniflora 32 24 Kondo 1973 16 32 Peng and Kenton 1982 32 Conran et al. 2002

Table 2. Comparison in karyotypes of two species in Byblis Total Average Chromosome Extremes of chromosome chromosome length from the Species Ploid level Stage arm ratios of length length largest to the chromosomes (µm) (µm) smallest B.filifolia 2x (2n =16) Early-metaphase 40.5 2.5 3.8-1.9 1.0-1.6 Mid-metaphase 23.3 1.5 1.7-1.1 1.0-1.5 B.liniflora 4x (4n =32) Early-metaphase 106.1 3.3 4.8-1.7 1.0-1.6 Mid-metaphase 54.0 1.7 2.7-1.2 1.0-1.6

metaphase chromosomes are shown in Figs. 1g-k, 2 and 3. were observed in all meristems analyzed. In B. filifolia, Features of prophase chromosomes between two species total chromosome length at early-metaphase was 40.5 µm, were karyomorphologcally same. Their chromosomes while total chromosome length at mid-metaphase was formed the heterochromatic segments at pericentromeric 23.3 µm. In contrast to B. filifolia, B. liniflora showed to- regions. In condensation behavior from prophase to tal chromosome length of 106.1 µm at early-metaphase, metaphase, most chromosomes of both species had early and 54.0 µm at mid-metaphase. To compare to the same condensing segments at the proximal regions. Moreover, stage of metaphase, the total length in B. liniflora was the prometaphase and early-metaphase chromosomes ob- nearly twice as long as that of B. filifolia. The metaphase served except for some small sized chromosomes had de- chromosomes showed a gradual decrease in size from the condenced segments at the distal regions in both arms largest to the smallest in both species. Two sat-chromo- (Fig. 1). During our investigation, it is quite rare to ob- somes, which were landmark in metaphase cells, were tain mid-metaphase cells with well-spread chromosomes, observed in both species. because all chromosomes at mid-metaphase stage were Chromosome lengths at early- and mid-metaphase in quite sticky nature, but not earlier stage of metaphase and two species are shown in Table 2. Byblis filifolia showed prophase. average chromosome lengths of 2.5 µm at early-meta- The chromosome numbers of two Byblis species phase, and 1.5 µm at mid-metaphase, and had 3.8 µm studied here are shown in Tables 1 and 2. The chromo- and 1.7 µm in the largest chromosome lengths at early- some counts in both species were made at prophase to metaphase and mid-metaphase, respectively. The small- metaphase stages. Byblis filifolia and B. liniflora had the est chromosome lengths were 1.9 µm and 1.1 µm at ear- diploidal and the tetraploidal chromosome numbers of ly-metaphase and mid-metaphase, respectively. These 2n=16 and 32, respectively. observed values gave extension rate of early-metaphase The same somatic complements with 16 chromo- chromosomes in B. filifolia: The largest chromosome at somes in B. filifolia and 32 chromosomes in B. liniflora early-metaphase was twice as large as that of mid-meta- 42 HOSHI ET AL CHROMOSOME STUDY IN BYBLIS 43 phase, while the smallest length at early-metaphase was Neither tetraploid chromosome number of B. filifolia 1.5 times as large as that of mid-metaphase. On the other nor diploid chromosome number of B. liniflora sensu hand, B. liniflora showed average chromosome lengths stricto has been reported. Although further chromosome of 3.3 µm at early-metaphase, and 1.7 µm at mid-meta- study is necessary to see intraspecific differentiation with phase, and had 4.8 µm and 2.7 µm in the largest chromo- euploid chromosome variation, chromosome counting some lengths at early-metaphase and mid-metaphase, re- seems to be good item to discriminate between these two spectively. The smallest chromosome lengths of B. species until now. liniflora were 1.9 µm and 1.2 µm at early-metaphase and Recent researches in somatic chromosomes in Byblis mid-metaphase, respectively. Thus, the largest length at have shown that the tetraploid species might be generated early-metaphase in B. liniflora was 2.5 times as large as by chromosome doubling with the diploid ancestor or that of mid-metaphase, while the smallest length at early- closely related species to B. liniflora. On the other hand, metaphase was 2.3 times as large as that of mid-meta- meiotic behavior supported that the tetraploid might not phase. The result indicated that the largest chromosomes be autoploid, because autoploid species generally has in the diploidal and the tetraploidal Byblis species had multivalent chromosomes. Since regular 16 bivalent higher chromosomal extending rates than those of the chromosomes were obtained in meiotic metaphase I of B. smallest chromosomes. liniflora sensu stricto (Peng and Kenton 1982), the tetra- ploid might be originated from allotetraploid or amphi- DISCUSSION diploid. Thus, mitotic and meiotic investigations lead us to make a hypothesis that at least two types of genome The present chromosome counts showed that the basic are exist in the B. liniflora complex. Moreover, there is a chromosome number was x=8 in both species, and chro- current report of the fossil newly recognized as a seed of mosome ploidies in somatic cells were the diploid in B. group in Byblis. This fossil was different from perennial filifolia and the tetraploid in B. liniflora. This means that B. gigantea, and was similar to annual species in the B. the B. liniflora complex contains euploid plants with dif- liniflora complex, but not B. aquatica which was only ferent ploidy levels. The chromosome counts of 2n=16 the exception in this complex. had the and 2n=32 were previously reported by Peng and Kenton basic chromosome number of x=9, while B. filifolia has (1982) and Conran et al. (2002). The present result was basic chromosome number of x=8. same to that of the previous reports. The last cytogeneti- Considering the species with primitive characters, it cal report mentioned that all species segregated by Low- was noted ascertained facts and plant-cytogenetically rie and Conran (1998) were the diploids with 2n=16 universal status as follows: There is no record of the tet- (Conran et al. 2002). At present the record of the tetra- raploid with basic chromosome number of x=9 ploid was only in B. liniflora sensu stricto in this genus. (2n=4x=36). A tetraploid is occasionally generated by There are some reports for taxonomical treatment and chromosome doubling event in diploid species. In the six speciation between B. filifolia and B. liniflora. Although species of Byblis, all species, except for B. liniflora sensu B. filifolia was described in the 1800’s (Planchon 1848), stricto, could be the diploids. Fossil seed of Byblis was not much importance had been attached to this distinc- similar to the present live seeds of the B. liniflora com- tion, because morphological characters of the species plex. Perennial species is generally more primitive than were similar to B. liniflora. Thus, B. filifolia was treated annual species [Smith (1967) in Dickison 1974]. There- as a synonym of B. liniflora for a long time. A taxonomi- fore, we speculate that B. filifolia or B. rorida might be cal examination of type specimens in Northwestern Aus- the more advanced and B. gigantea might be primitive tralia by Conran and Lowrie (1993) summarized a new within the genus. On the basis of this speculation, x=9 proposal to describe two subspecies of B. liniflora subsp. could be the primitive basic chromosome number. It liniflora and B. liniflora subsp. occidentalis within B. might occur during evolutional progress toward the an- liniflora. Byblis liniflora subsp. occidentalis grew under nual species by losing one basic chromosome number to generally drier and better drained conditions than B. lini- establish x=8. flora subsp. liniflora did. Byblis liniflora subsp. occiden- Although predicting of chromosomal doubling event talis had more flowers at the stem apex, larger petals with easily come to us after karyotype compared between the serrate apices, and the elongate anthers longer than their two subspecies of the B. liniflora complex, the same filaments thanB. liniflora subsp. linifloradid. Geographi- numbers of sat-chromosomes in the diploid and the tetra- cal and morphological descriptions of the two subspecies ploid species were nonadditive. It was speculated that the led to a review on taxonomical works with the previous nonadditivity of sat-chromosome number in the tetra- record of B. filifolia, since the record was similar to that ploid plant might be due to suppression or deleting after of B. liniflora subsp. occidentalis. Currently, B. filifolia chromosome doubling. has been treated as well-defined species of Byblis, while Further studies by using of FISH and GISH tech- B. liniflora subsp. occidentalis has been treated as syn- niques are necessary to clarify karyotype evolution and onym of this species (Lowrie and Conran 1998). species relationships of Byblis. 42 HOSHI ET AL CHROMOSOME STUDY IN BYBLIS 43

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