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© 2015 The Japan Mendel Society Cytologia 80(2): 151–157

Fluorescent Band Pattern of Chromosomes in amabilis,

Masahiro Hizume*

Faculty of Education, Ehime University, 3 Bunkyo-cho, Matsuyama, Ehime 790–8577, Japan

Received October 27, 2014; accepted November 18, 2014

Summary belongs to one of three monotypic genera in Pinaceae. This had 2n=44 chromosomes in somatic cells and its karyotype was composed of four long submetacentric chromosomes and 40 short telocentric chromosomes that varied gradually in length, supporting previous reports by conventional staining. The chromosomes were stained sequentially

with the fluorochromes, chromomycin A3 (CMA) and 4′,6-diamidino-2-phenylindole (DAPI). CMA- bands appeared on 12 chromosomes at near terminal region and proximal region. DAPI-bands appeared at centromeric terminal regions of all 40 telocentric chromosomes. The fluorescent-banded karyotype of this species was compared with those of other Pinaceae genera considering taxonomical treatment and molecular phylogenetic analyses reported. On the basis of the fluorescent-banded karyotype, the relationship between Pseudolarix amabilis and other Pinaceae genera was discussed.

Key words Chromomycin, Chromosome, DAPI, Fluorescent banding, Pinaceae, Pseudolarix amabilis.

In Pinaceae, 11 genera with about 220 species are distinguished and grow mostly in the Northern Hemisphere (Farjon 1990). Most genera are , and only Larix and Pseudolarix are . Pinus is the largest in species number, and , and Pseudolarix are monotypic genera. The of Pinaceae with 11 genera is complicated, having some problems in species or variety level. Several higher taxonomic treatments were reported on the base of anatomy and morphology such as canal in the vascular cylinder, scale, position of mature cones, male strobili in clusters from a single , and molecular characters in base sequences of several DNA regions. The family Pinaceae was divided into three subfamilies; Pinoideae (Pinus), Lariciodeae (, Larix, Pseudolarix) and Abietoideae (Abies, Cathaya, , Picea Psudotsuga, ) by Pilger (1926), into two groups of Abies-Cedrus- Keteleeria-Pseudolarix-Tsuga and Cathaya-Larix-Picea-Pinus- by Melchior and Werdermann (1954), and into four subfamilies: Pinoideae (Pinus), Piceoideae (Picea), Laricoideae (Larix, Cathaya, Pseudotsuga), and Abietoideae (Abies, Cedrus, Pseudolarix, Keteleeria, Nothotsuga, Tsuga) by Hart (1987) and Frankis (1989). These classifications should be referred to Farjon (1990). Early molecular phylogenetic studies using PCR-PFLP and base sequence of rbcL (Tsumura et al. 1995, Chaw et al. 1997) indicated two groups in the phylogenetic but the trees had low reliability. Wu and Hu (1997) and Wang et al. (2000) presented three groups discussing relationships between or among genera on the basis of various morphology and anatomical structures. Recently, Gernandt et al. (2008) used a matrix of morphology, anatomical characters and base sequences of matK and rbcL and constructed reliable phylogenetic trees, which indicated that Pinaceae is divided two subfamilies, Abietoideae (Cedrus, Abies, Keteleeria, Pseudolarix, Nothotsuga, Tsuga) and Pinoideae (Larix, Psudotsuga, Cathaya, Picea, Pinus). The molecular

* Corresponding author, e-mail: [email protected] DOI: 10.1508/cytologia.80.151 152 M. Hizume Cytologia 80(2) phylogenetic tree suggests that Pseudolarix is put in subfamily Abietoideae and relates to Tsuga and Nothotsuga. Most genera of Pinaceae are studied on basic chromosome number and their karyological information are deposited. Ten genera have common basic chromosome number, x=12 except for the monotypic genus, Pseudolarix possessing n=22 and 2n=44 and one species of Pseudotsuga, Pt. menziesii having n=13 and 2n=26 (Sax and Sax 1933, Khoshoo 1959, Mehra 1968, Hizume 1988). Karyotypes of species are common in each genus with variation in number or location of secondary constriction and slight change of chromosome shape. Karyotypes of the genera in Pinaceae were divided into several groups (Hizume 1988, Li 1995). Nkonggolo and Mehes-Smith (2012) reviewed recent molecular cytogenetic studies on Pinaceae karyotype implicating with molecular phylogeny. In Pinaceae most chromosomes are meta- and submeta-centric. Only two species, Pt. menziesii and Pl. amabilis, have telocentric chromosomes in their chromosome complements. Although five other Pseudotsuga species have 2n=24 chromosome number in common with Pinaceae genera and their karyotypes composed of 12 long metacentric chromosomes and 12 short submetacentric chromosomes, Pt. menziesii has 2n=26 chromosomes composed of 10 long metacentric chromosomes, 12 short submetacentrics and four short telocentrics. Barner and Christiansen (1962), Thomas and Ching (1968), El-Kassaby et al. (1983) and Sziklai et al. (1987) speculated that four telocentric chromosomes were derived from two pairs of long metacentric chromosomes by centromeric fission. Until now the explanation is not demonstrated cytogenetically by making a hybrid between 2n=26 and 2n=24 species and the observation of the meiotic configuration in the pollen mother cells of the hybrid. The karyotype (2n=44) of Pl. amabilis is composed of two pairs of submetacentric chromosomes and 20 pairs of telocentric chromosomes, which was explained with polyploidy and chromosome reduction (Sax and Sax 1933) and centromeric fission in the karyotype of certain Pinaceae genus (Khoshoo 1959, Mergen 1961, Gustafsson and Mergen 1964, Li 1994). Recently Zonneveld (2012) reported genome seizes in 172 species including five genera of subfamily Abietoideae, 32–52 pg/C in Abies, 31–41 pg/C in Tsuga, 48.4 pg/C in Keteleeria, 32–40 pg/C in Cedrus, and 52.2 pg/C in Pseudolarix. The deviation of genome sizes suggests that Pl. amabilis might be diploid species. This study reports fluorescent banding pattern of chromosomes in Pl. amabilis and discusses its origin and phylogenetic position in Pinaceae.

Materials and methods

Small trees of Pseudolarix amabilis (Nelson) Rehder were obtained from commercial sources and planted in a pot. In the spring, growing root-tips were collected and treated in 2 mM 8-hydroxiquinoline or 0.05% colchicine for 6–7 h. Then the root-tips were immersed in a fixative (ethanol : acetic acid : chloroform=2 : 1 : 1) and stored in a freezer. Fixed root-tips were through 70% ethanol and immersed in water. Then the root-tips were soaked in 45% acetic acid for 5 min and transferred into 45% acetic acid at 60°C for 10 min. The root-tips were transferred into cold 45% acetic acid. Under a stereomicroscope, each root-tip was dissected by tweezers and a needle, and then the meristematic tissue was scooped and put on a glass slide. An aliquot of 45% acetic acid was dropped on the tissue and a cover glass was put on it. Cells of meristems were spread and squashed. The preparation was put on dry ice for a few minutes and the cover glass was ripped off. The preparation was air-dried overnight. Sequential fluorescent banding method with guanine specific CMA and adenine-thymine specific DAPI was described earlier by Kondo and Hizume (1982). The dried preparation was immersed into McIlvaine buffer pH 7.0 for 30 min. The glass slide was treated with 0.1 mg/mL distamycin A for 10 min, then washed with the buffer containing

5 mM MgSO4 for 10 min and stained with 0.1 mg/mL CMA in the buffer. After a wash with the buffer for 10 min the preparation was mounted with non-fluorescence glycerin and stored in a 2015 Fluorescent Band Pattern of Chromosomes in Pseudolarix amabilis, Pinaceae 153 refrigerator at 4°C for more that one night. After storage the CMA-stained preparation was observed under an epifluorescence microscope using a B filter cassette. Then the preparation was dipped in distilled water until the cover glass dropped off. Then the preparations were treated with acetic–alcohol (3 : 1) to remove CMA and glycerin, rinsed briefly with distilled water and then air- dried. The preparation was put in the buffer without MgSO4 for 10 min, treated with 0.1 mg/mL actinomycin D for 10 min and then washed again for 10 min with the buffer. The preparation was stained with 0.1 μg/mL DAPI for 5 min then washed with the buffer for 5 min. After mounting with the buffer–glycerin (1 : 1, v/v) mixture the same chromosomes observed with CMA were observed under the fluorescence microscope using a UV filter cassette. Fluorescence photographs of the same chromosomes stained with CMA and DAPI were taken on a film (TMAX, Kodak) and developed with double dilution of D-76.

Results and discussion

The chromosome number in somatic cells was observed to be 2n=44 in all six of Pl. amabilis examined. The chromosome complement was composed of four long submetacentric chromosomes and 40 short telocentric chromosomes, gradually decreasing in their lengths (Fig. 1). The somatic chromosome number and the karyotype observed were similar to previous studies (Sax and Sax 1933, Mergen 1961, Hizume 1988, Li 1994). After CMA-banding, 12 CMA-bands appeared in the somatic chromosome complement and most CMA-bands appeared at near region of centromeric ends (Fig. 1A). Ten CMA-bands were located at the distal position of DAPI-bands at the terminal or centromeric region, but not the terminal end of telocentric chromosomes. Two long submetacentric chromosomes had a somewhat weak CMA-band at the proximal region of their long arm. DAPI-bands appeared in all 40 telocentric chromosomes and were located at the centromeric or terminal region of telocentric chromosomes. Four long submetacentric chromosomes did not have any DAPI-bands (Fig. 1B). When the CMA-banding pattern was compared with the DAPI-banding pattern, the CMA-band was DAPI-negative and the DAPI-band was CMA-negative, same in other Pinaceae species and other

Fig. 1. Fluorescent-banded somatic chromosomes of Pseudolarix amabilis at metaphase. (A) CMA staining. (B) DAPI staining. Bar=5 μm. 154 M. Hizume Cytologia 80(2) species. The karyotype and fluorescent banding pattern of Pl. amabilis was quite different from those of other Pinaceae genera reported. In Pinaceae karyotypes revealed by conventional staining have been described already in nearly all genera (Sax and Sax 1933, Hizume 1988, Chu and Sun 1981) except for Nothotsuga. Among Pinaceae genera the karyotype (2n=44) of Pseudolarix composed of 40 telocentric chromosomes and four submetacentric chromosomes was very different from that (2n=24) of other genera. The unique karyotype of Pseudolarix has been speculated to be generated by centromeric fission from 20 meta- and/or submeta-centric chromosomes of karyotype in some other genera (Mergen 1961, Gustafsson and Mergen 1964, Li 1995). The speculation was postulated just by comparison of conventional karyotypes and not by any cytogenetic or molecular evidence. Among several chromosome banding methods, fluorescent banding is an exquisite technique for karyotype analysis in Pinaceae species in that it has simple and easy procedures and produces highly reproducible banding patterns. The CMA- and DAPI-banding techniques were applied first to chromosomes of Pinus species in Pinaceae (Hizume et al. 1983). In Pinus species many fluorescent bands appeared on all chromosomes and the CMA- and DAPI-banding patterns were useful for chromosome identification and karyological relationship among certain species (Hizume et al. 1983, 1989b, 1990). The fluorescent banding patterns of Pinaceae species were revealed and deposited in Abies (Shibata et al. 2004, Puizina et al. 2008), Cedrus (Dagher-Kharrat et al. 2001), Keteleeria (Hizume et al. 1993b), Larix (Hizume and Tanaka 1990, Hizume et al. 1988, 1993a, 1994, 1995, 1998, 2002), Picea (Hizume et al. 1989a, 1991, Hizume and Kuzukawa 1995), Pinus (Hizume et al. 1983, 1989b, 1990), Psudotsuga (Hizume and Kondo 1992, Hizume and Akiyama 1992, Hizume et al. 1996). Three genera, Tsuga, Nothotsuga and Cathaya, were not examined yet by fluorescent banding method. In most species of Pinaceae genera several interstitial CMA-bands on chromosomes appeared on secondary constriction or sites of 45S rDNA by ISH and some thin CMA-band was localized sites of 5S rDNA (Hizume and Kuzukawa 1995, Hizume et al. 1995, 1996, Shibata and Hizume 2008, Nkongolo and Mehes-Smith 2012). Only Pinus species had CMA-bands at centromeric region of certain chromosomes in addition to interstitial regions (Hizume et al. 1983, 1989b, 1990). The DNA (PDCD501 and PDCD159) was cloned directly from the dissection of centromeric chromosome fragments in Pinus densiflora and PDCD501 was localized on centromeric CMA- band revealed by FISH (Hizume et al. 2001). Pseudolarix have 10 CMA-bands at proximally interstitial region and two weak CMA-bands at proximal region of long arm of the submetacentric chromosomes (Fig. 1A) The near-centromeric localization of CMA-bands in telocentric chromosomes is very unique in the Pinaceae genera. Previous karyotype analyses of Pseudolarix showed secondary constrictions and small constrictions at proximally interstitial region of two or more pairs of telocentric chromosomes (Hizume 1988, Li 1994). To reveal that some or all of the 12 CMA-bands of Pseudolarix had NOR function or rDNA, chromosomes of Pseudolarix should be analyzed by FISH using 45S and 5S rDNA probes. Some genera of Pinaceae had DAPI-bands at proximal and/or interstitial chromosome regions. Pinus species had centromeric DAPI-bands on some chromosomes and many interstitial thin DAPI- bands in all chromosomes (Hizume et al. 1983, 1989b, 1990), Larix species have DAPI-band at proximal region of single, not both, arm of most chromosomes (Hizume and Tanaka 1990, Hizume et al. 1993b, 1994, 1995, 1998, Liu et al. 2007), Cedrus species had DAPI-bands at centromeric regions of most chromosomes (Dagher-Kharrat et al. 2001) and had four chromosomes with centromeric DAPI-bands (Puizina et al. 2008). It was proposed that Pseudolarix karyotype was generated by centromeric fission from Larix (Mergen 1961, Gustafsson and Mergen 1964) or Tsuga (Li 1994). Larix has many chromosomes with DAPI-band at proximal region of one chromosome arm. When the chromosome brakeage occurred at centromere into telocentric chromosomes, about half of the telocentric chromosomes 2015 Fluorescent Band Pattern of Chromosomes in Pseudolarix amabilis, Pinaceae 155 had DAPI-bands and the residual chromosomes had no DAPI-bands. The DAPI-band pattern generated from Larix by centromeric fission coincided with that of Pseudolarix. Also, Larix is taxonomically treated and put into different subfamily Pinoideae in consensus classification of Pinaceae. The phenomenon seems to indicate that Larix is not a sister genus or ancestor of Pseudolarix. In Larix leptolepis, AT-rich DNA (LPD) was cloned and sequenced, and repeated in tandem. The LPD was localized at DAPI-bands by FISH (Hizume et al. 2002). PCR products were sequenced in nine species and four varieties of Larix examined. Their sequences were very similar to each other. Southern blotting was used as a probe and was preliminarily applied on 14 Pinaceae species and four species, Abies firma, Picea gmelini, Pinus grabra and , showing weak redder signals. LPD might distribute regardless of the amount of LPD around Pinaceae. The analyses of PCR using the primer of LPD, and FISH and Sothern hybridization with probe of LPD in genome of Pseudolarix and other genera might reveal distribution of LPD in genemes of Pinaceae. Another possible approach is to clone the AT-rich repeat from Pseudolarix, Cedrus and other genera having proximal DAPI-band of Abietoideae, and compare it with their sequences and chromosomal localization. Larix and Psudotsuga have very similar or the same karyotype (Hizume 1988) and similar CMA-band pattern and 5S and 45S rDNA sites (Hizume and Akiyama 1992, Hizume et al. 1995, 1996, Liu et al. 2007, Goryachkina et al. 2013). They were put in subfamily Pinoideae and into the same clade by many morphological and molecular phylogeny trees (Wang et al.1998, Gernandt and Liston 1999, Gernandt et al. 2008). Pseudotsuga did not have any Larix- type proximal DAPI-bands or a detectable amount of LPD sequence. This indicates that after separation of these genera LPD generated and amplified in tandem very quickly at proximal region of Larix chromosomes. In subfamily Abietoideae four species of Cedrus had centromeric DAPI-bands on 11 or 12 pairs of chromosomes (Dagher-Kharrat et al. 2001). Chromosome breakage occurred at the centromeric regions of 20 meta- and/or submeta-centric chromosomes having DAPI-bands to generate 40 telocentric chromosomes with proximal DAPI-band. The possibility that Pseudolarix was differentiated from Cedrus was conceived from comparison of just DAPI-banding patterns. To determine the closely related genus to Pseudolarix, the AT-rich sequence located at the centromeric DAPI-bands should be analyzed in Pseudolarix, Cedrus and Tsuga by cloning of the AT-rich repeat and fluorescent banding. On the basis of comparison of DAPI-band patterns, Cedrus species seems a tentative ancestor of Pseudolarix but the DAPI-band composed of AT-rich repeat should not be ignored to generate quickly as observed in Larix. The comparison of CMA-band patterns of two genera, Pseudolarix and Cedrus, showed very different CMA-band patterns. The CMA-band pattern of Pseudolarix was difficult to be originated from that of Cedrus. A tentative candidate for the ancestor of Pseudolarix is proposed to be Cedrus with problems of CMA-banding pattern. Molecular phylogenetic studies (Wang et al. 2000, Gernandt et al. 2008) put Tsuga at a position close to Pseudolarix, Tsuga should be analyzed by the fluorescent banding and molecular cytogenetic analyses. More molecular cytogenetic studies are needed for a complete understanding of the phylogenetic relationship of extant species and genera in Pinaceae.

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