Chromosome Botany (2017)12(2): 17-32 ©Copyright 2017 by the International Society of Chromosome Botany

Chromosome banding in Picea IV. Comparative karyotype analysis of fluorescent band patterns in 25 taxa

Masahiro Hizume

Faculty of Education, Ehime University, Matsuyama 790-8577 Japan

Author for correspondence: ([email protected]) Received August 1, 2017; accepted August 18, 2017

ABSTRACT: In 25 taxa of Picea the somatic chromosomes were analyzed by a fluorescent banding technique using chromomycin A3 (CMA) and DAPI. They had 2n=24 chromosome number and similar karyotypes among . On metaphase chromosomes in each taxon the CMA-bands appeared at the interstitial and centromeric regions. DAPI-bands appeared on chromosomes of some species. The CMA-band pattern of each taxon was described after identification of most homologous chromosomes and compared among taxa. An alignment of species based on CMA-band pattern with some exception were coincident on their geographic distribution, and discussed on interspecific relationships, comparison to molecular phylogeny and DNA sequences corresponding to fluorescent bands.

KEYWORDS: Chromomycin, Chromosome, DAPI, Fluorescent banding, Karyotype, Phylogeny, Picea,

The genus Picea is one of genera in Pinaceae and coincident with secondary constrictions and 45S rDNA composed of 40-50species, and distributing North loci (Hizume and Kuzukawa 1995). The other progress Hemisphere (Farjon 1990, 2001, 2010; Lockwood et al. was isolation of tandem repeat (Sau3A) in P. glauca and 2013). The karyotype study by smear or squash methods localized at centromeric region of several chromosomes in had been started in several by Sax and Sax (1933). P. glauca and P. sitchensis (Brown et al. 1998). The FISH During next forty years the chromosome and karyotypes using probes of 45S rDNA, 5S rDNA and Sau3A applied were reported by conventional staining in many species of on 11 species showed presence of Sau3A in all species Picea (Mehra and Khoshoo 1956; Terasmaa 1971, 1975; genome examined and FISH pattern of three probes Liu and Li 1985; Hizume 1988; Nkongolo 1996; Inceer et supplied valuable information of their relationships among al. 2009). The studies revealed that Picea species have a Picea species (Shibata and Hizume 2008). The most similar karyotype composing of eight long metacentric appropriate procedure for handling many species, easy and chromosomes and four short chromosomes, and carrying reproducible seems the fluorescent staining. several secondary constrictions. Detail karyotype analysis This time 25 taxa of Picea were analyzed and revealed variation in the secondary constrictions between described on their florescence banding patterns, especially P. rubens and P. mariana (Nkongolo 1996) and that among CMA-bandings. The CMA-band patterns were compared populations in P. orientalis (Inceer et al. 2009). Resent among species to reveal relationships of taxa in Picea. The genome study constructed high density gene maps in P. relationship of CMA-band pattern was compared with abies, P. glauca, P. mariana × P. rubens and compared phylogenetic treatment and molecular phylogenetic with each other (Acher et al. 2004; Pelgas et al. 2006). analysis to reveal chromosomes change during phylogeny The linkage maps were similar among species suggesting or speciation in Picea. A longitudinal differentiation of strongly that structural change of chromosome such as chromosomes was also discussed. translocation was occur rarely. In a comparative chromosome study among Picea species this indicates that MATERIALS AND METHODS an identification of each homoeologas chromosome is Materials Seeds were collected in fields or supplied very important for understanding their real genetic from several institutes shown in Table 1. The seeds were a relationships. To identify homologous or homoeologous sterilized with 2% H2O2 for 10 min then washed with a chromosomes the modern cytological techniques such as sterilized water. The seeds were sown on a wet filter paper C-banding (Teoh and Rees 1977; Kohler et al. 1996), in Petri dishes. After 10-14 days root tips of primary roots fluorescent banding (Hizume et al. 1989a, 1991) and in with about 8 mm length were collected for cytological situ hybridization (Brown et al. 1993; Hizume and observation. Kuzukawa 1995; Hizume et al. 1999; Shibata and Hizume 2008) were applied to Picea chromosomes but not so Preparation of chromosome and fluorescent banding much. The CMA-banding revealed identification of most method The excised root tips were treated with 0.05% chromosomes and some variations intraspecific and colchicine at 20℃ for 8 h, then fixed in a fixative mixed between varieties (Hizume et al. 1989a, 1991). By in situ with ethanol and acetic acid (3:1) and stored in a freezer hybridization (ISH) interstitial CMA-bands were until use. The fixed root tips were washed with 70%

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Table 1. Locality or origin of seeds of each Picea taxon examined. Taxon Locality and Origin (L.) H. Karst. Viljandi, Estonia P. alcoquiana (Veitch ex Lindl.) Carrière Monbetsu, , Japan P. asperata Mast. Huichuangalinzhen, Weiyuaan, , P. crassifolia Kom. Datong，Qinghai, China P. engelmannii Parry ex Engelm. Bastion Mountain, British Columbia, Canada P. glauca (Moench) Voss Paddle Prairie, Alberta, Canada P. glehnii (F. Schmidt) Mast. Kwakami, Hokkaido, Japan P. jezoensis (Siebold & Zucc) Carrière var. jezoensis Monbetsu, Hokkaido, Japan P. jezoensis (Siebold & Zucc) Carrière Kiso, Nagano, Japan var. hondoensis (Mayr) Rehder P. koraiensis Nakai Heaobeihu，Ningan，Helongjiang, China P. koyamaeShiras. Suwa, Nagano, Japan P. koyamae Hayashi Yatsugatake, Obuchizawa, Yamanashi, Japan var. acicularis (Shiras. & Koyama) T. Shimizu P. likiangensis (Franch.) E. Pritz. Yunshping, Lijian, Yunnan, China P. mariana (Mill.) Britton & al. Indian bay,Newfoundland and Labrador, Canada P. maximowiczii Regel ex Mast. var. senanensis Hayashi Minamisaku, Nagano, Japan P. obovata Ledeb. Rudni, Altai Vostochro, Kazakhstan P. omorika (Ppnčić) Purk. Vranjak, Serbia P. orientalis (L.) Peterm. commercial source P. pungens Engelm. Gunnison. Colorado, USA P. rubens Sarg. Green river, New Brunswick, Canada P. schrenkiana Fisch. & C. A. Mey. North Tienshan, Alma-Ata, Kazakhstan P. sitchensis (Bong.) Carrière Gueen Charlotte Iland, British Columbia, Canada P. smithiana (Wall.) Boiss. Gyirong, Tibet P. torano (Siebold ex K. Koch) Koehne Fujinomiya,Yamanashi, Japan P. wilsonii Mast. Loutacang, Nanping, Sichuan, China

ethanol and distilled water, and finally soaked in 45% species we intended to observe and reliable karyotype by acetic acid. The root tips were treated in 45% acetic acid at the fluorescent banding. The banded karyotypes were 60℃ for 10 min and then transferred in cold 45% acetic identified homologous chromosome in each taxon and acid. A meristematic tissue was dissected by a needle with compared with homoeologous chromosomes among taxa. flat tip under a stereomicroscope. The meristematic cells Nine chromosome pairs were easily identified in each were spread in 45% acetic acid and pressed on a glass chromosome compliment of all taxa. Therefore the slide under a coverslip. The glass slides were put on a CMA-band patterns were compared in each identified pair dry-ice block for 2 min and then cover glasses were and the other four pairs were compared together. The removed from the slides. The slides were air dried nomenclature of chromosomes of Picea species follows overnight. The fluorescent banding procedures were Hizume et al. (1991). Below nomenclature system of described previously (Hizume et al. 1983, 1988, 1989a). chromosome pairs using chromosome shape and location The figures of fluorescent banded chromosomes were of interstitial CMA-band was used. recorded on a sensitive film (T-max, Kodak). Lm1: the longest metacentric chromosome Description of karyotype and fluorescent banded Lm2: a long metacentric chromosome with interstitial chromosomes Many conventional karyotype analyses CMA-band accompanying thin CMA-band at its close in Picea species revealed that the karyotype was proximal side composed of 16 long metacentric chromosomes and eight Lm3: a long metacentric chromosome with CMA-band at short submeta- and meta-centric chromosomes (Mehra and terminal interstitial region Khoshoo1956; Liu and Li 1985; Hizume 1988; Siljak- Lm4: a long metacentric chromosome with CMA-band at Yakovlev et al. 2002). The secondary constrictions proximally medium region of its arm appeared at an interstitial region of several chromosomes, Lm5: a long somewhat heterobrachial metacentric and their number and location varied among or chromosome with interstitial CMA-band on long arm species or studies (Terasmaa 1975; Liu and Li 1985; Lm6-8: long metacentric chromosomes with or without Hizume 1988; Nkongolo 1996). The chromosome interstitial CMA-band depending on species. The arrangement in karyotype varies among studies by chromosomes pairs can identify in each species but different authors. In order to compare karyotype among beyond species identification of these chromosome pairs

FLUORESCENT CHROMOSOME BANDING IN PICEA 19

were difficult. This time these chromosomes were proximal side of them. Lm3 chromosome had an treated together. interstitial CMA-band. Lm4 and Lm5 chromosomes had Ssm1: the longest submetacentric chromosome CMA-bands at interstitial and centromeric regions. One Sm1: a short metacentric chromosome Lm had no CMA-band. Short submetacentric Sm2: a short metacentric chromosome more symmetric chromosomes (Ssm1) had centromeric CMA-band and than Sm1 short metacentric (Sm1) had CMA-bands at interstitial Ssm2: the shortest chromosomes having centromere at region of short arm and centromeric region. Other short the asymmetric position. chromosomes (Sm2, Ssm2) had no CMA-band. After Small B-chromosomes were recognized two types and DAPI-staining bright DAPI-band did not observed. they were described metacentric and submetacentric Negative DAPI-bands appeared at all regions of interstitial B-chromosomes as Bm and Bsm, respectively. and centromeric CMA-bands and weak DAPI-bands appeared at interstitial region of some chromosomes RESULTS (Fig.1B). All plants of taxa examined had 2n = 24 chromosomes and In P. alcoquinata four interstitial and five centromeric sometimes few small B-chromosomes. Karyotypes of taxa CMA-bands appeared in the karyotype (Fig. 2A). Lm1 and were similar each other and supported karyotypes two Lm chromosomes had CMA-band only in centromeric previously reported. The CMA-bands appeared at an region. Lm3, Lm4 and Lm5 chromosomes had interstitial region and/or centromeric region. In most taxa CMA-bands at both interstitial and centromeric regions. homologous chromosomes were identified by CMA-band Lm2 and Sm1 chromosomes had interstitial CMA-band pattern. Among species nine chromosome pairs were and, two Lm and Ssm1 chromosomes had centromeric referable and other pairs were not. DAPI-bands appeared CMA-bands. Several DAPI-bands were observed at at the interstitial region without CMA-band and centromeric region (Fig. 2B). centromeric region in several species, such as P. mariana (Fig. 14). The analysis of DAPI-band pattern seemed hard, then in this paper the CMA-band pattern of karyotype was described mainly and compared among taxa.

Fluorescent banding pattern of chromosomes in each Picea taxon In P. abies six interstitial CMA-bands appeared clearly in the karyotype, and one and four centromeric CMA-bands appeared clear and weak, respectively (Fig.1A). The longest metacentric chromosomes (Lm1) and other one long metacentric chromosome (Lm) had no CMA-band. Lm2 chromosome had an interstitial CMA-band and thin CMA-band accompanied at the

Fig. 2. Fluorescent banded chromosomes in Picea alcoquinata. A: CMA-banding and B: DAPI-banding. Bar=10μm.

In P. asperata the Lm2, Lm3, Lm4 and one Lm chromosomes had an interstitial CMA-band on one arm (Fig. 3A). Ssm1chromosome had a centromeric CMA- band and Lm4 and Ssm1chromosomes did a weak CMA-band at centromeric region. The Lm1 and the other three Lm chromosomes, and Sm1, Sm2 and Ssm2 chromosomes had no CMA-band. After DAPI-banding the regions of CMA-bands were dull on bright chromosome arms. Few faint DAPI-bands appeared at the interstitial region of some chromosomes (Fig. 3B). In P. crassifolia four interstitial CMA-bands appeared in the Lm2-4 and one Lm chromosomes, and one centromeric CMA-band did on Ssm1 chromosome (Fig. 4A). In some plants Sm1 chromosomes had weak CMA-band at interstitial region of short arm (Fig. 4B). Fig. 1. Fluorescent banded chromosomes in Picea abies. Small B-chromosomes (Bm and Bsm) appeared in some A: CMA-banding and B: DAPI-banding. Bar=10μm. plants.

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Fig. 3. Fluorescent banded chromosomes in Picea asperata. A: CMA-banding and B: DAPI banding. Fig. 5. Fluorescent banded chromosomes in Picea Bar=10μm. engelmannii. A: CMA -banding and B: DAPI -banding. Bar=10μm.

One Lm had interstitial CMA-band and Ssm1 had a centromeric CMA-band. Centromeric CMA-band of Lm4 varied among plants. Other three (one Lm, Sm2 and Ssm2) had not CMA-band. The regions of CMA-bands appeared darker than chromosome arms after DAPI-staining and few faint DAPI-bands appeared at interstitial regions of some chromosomes (Fig. 6B). Metacentric B-chromosome (Bm) had complex CMA- band pattern.

Fig. 4. Fluorescent banded chromosomes in Picea crassifolia. A: CMA-banding and B: CMA-band. Bar=10μm.

In P. engelmannii six interstitial and eight centromeric CMA-bands appeared in karyotype (Fig. 5A). Lm1, one Lm and Ssm1 chromosomes had only centromeric CMA-bands. Lm2 chromosomes had an interstitial CMA-band. Lm3, Lm4, two Lm and Ssm1 chromosomes had CMA-bands at both interstitial and centromeric regions. One Lm chromosome and Sm2 chromosome had no CMA-band. Clear DAPI-band appeared at centromeric region of the shortest Ssm2 chromosomes. Faint interstitial DAPI-bands were also observed. The CMA-banded regions were darker than chromosome arms excepting for centromeric CMA-band region of Ssm2 displaying DAPI-band. Several weak DAPI-bands appeared at Fig. 6. Fluorescent banded chromosomes in Picea interstitial regions of some chromosomes (Fig. 5B). glauca. A: CMA-banding and B: DAPI-banding. In P. glauca seven interstitial CMA-bands and, six Bar=10μm. clear and two thin centromeric CMA-bands appeared in the karyotype (Fig. 6A). The Lm1 chromosomes had a In P. glehnii six interstitial and five centromeric centromeric CMA-band. Lm2 had a interstitial CMA-band. CMA-bands were observed (Fig.7A). Four centromeric Lm3, Lm4, two Lm and Sm1 chromosomes had bands were weak. Lm3, Lm4 and one Lm chromosomes CMA-bands at both interstitial and centromeric regions.

FLUORESCENT CHROMOSOME BANDING IN PICEA 21 had CMA-bands on both interstitial and centromeric regions. Lm2, one Lm and Sm1 chromosomes had interstitial CMA-band and, one Lm and Ssm1 had centromeric CMA-band. Lm1, one Lm, Sm2 and Ssm2 chromosomes had not CMA-band. Dark region appeared at CMA-bands after DAPI-staining (Fig.7B).

Fig. 9. Fluorescent banded chromosomes in var. hondoensis. A: CMA-banding and B: DAPI-banding. Bar=10μm. Fig. 7. Fluorescent banded chromosomes in Picea

glehnii. A: CMA-banding and B: DAPI-banding. Bar=10μm. reported by Hizume and Kuzukawa (1996). In P. koraiensis five interstitial and one centromeric CMA-bands appeared in the karyotype (Fig. 10A). Lm2, In P. jezoensis var. jezoensis six interstitial and four Lm3, Lm 4 and two Lm chromosomes had a interstitial centromeric CMA-bands appeared in karyotype (Fig. 8A). CMA band. Ssm1 chromosome had weak CMA-band at Interstitial CMA-bands appeared on Lm2-4, one Lm and proximal region of the short arm. Other chromosomes had Sm1 chromosomes. Centromeric CMA-bands appeared on no CMA-band. After DAPI-staining chromosome arms Lm4 and two Lm. Lm4 and one Lm chromosomes had fluoresced homogeneously having negative band at CMA-bands at both interstitial and centromeric regions. regions of CMA-band (Fig. 10B). Negative DAPI-band appeared at the region of CMA-bands. Interstitial DAPI-bands appeared on several chromosomes (Fig. 8B). In P jezoensis var. hondoensis (Fig. 9) CMA band pattern of var. hondoensis was very similar to that of var. jezoensis. Difference was observed in Sm1 chromosome without interstitial CMA-band as

Fig. 10. Fluorescent banded chromosomes in Picea koraiensis. A: CMA-banding and B: DAPI-banding. Bar=10μm.

In P. koyamae five interstitial and no centromeric Fig. 8. Fluorescent banded chromosomes in Picea CMA-bands appeared in the karyotype (Fig. 11A). Two jezoensis var. jezoensis. A: CMA-banding and B: Lm chromosomes had two CMA-bands on interstitial and DAPI-banding. Bar=10μm. centromeric regions. Lm2, Lm3, Lm4 and Lm5

22 HIZUME chromosomes had interstitial CMA-band, and Sm1 arms. Four chromosomes (Lm3, Lm4, one Lm and Sm1) chromosomes had weak interstitial CMA-band. Lm1, two had interstitial CMA-band. Other seven chromosomes had Lms, Sm2 and Ssm2 chromosomes had no CMA-band. not CMA-band. Thin DAPI-bands appeared at interstitial Few weak interstitial DAPI-bands appeared on several region of several chromosomes. The region of CMA-band chromosomes. Metacentric B-chromosomes having DAPI- and centromeric regions of some chromosomes were band at the proximal region were observed (Fig. 11B). The negative DAPI-bands indicating some centromeric region CMA-band pattern of P. koyamae var. acicularis (Fig. contained AT-rich sequence (Fig. 13B). Submetacentric 12A) was same to that of P. koyamae. Thin interstitial B-chromosome had DAPI-positive long arm. DAPI-bands appeared on several chromosomes (Fig. 12B). A metacentric B-chromosome with proximal DAPI-band appeared.

Fig. 13. Fluorescent banded chromosomes in Picea Fig. 11. Fluorescent banded chromosomes in Picea likiangensis. A: CMA-banding and B: DAPI-banding. koyamae. A: CMA-banding and B: DAPI-banding. Bar=10μm. Bar=10μm. In P. mariana six interstitial CMA-bands and eight centromeric CMA-bands were observed in the karyotype (Fig. 14A). The chromosomes with both interstitial and centromeric CMA-bands were dominant in this species. The chromosomes of this CMA-band pattern were Lm2-4, two Lm and Sm1. Two chromosomes (one Lm and Ssm1) had CMA-band at centromeric region. The other four chromosomes had no CMA-band. Many DAPI-bands appeared at centromeric region of eight long metacentric chromosomes and thin DAPI-bands did at interstitial regions in several chromosomes (Fig. 14B). Some centromeric CMA-bands showed DAPI-positive or – negative in depend on chromosomes for example of stars in Fig. 14. Six centromeric regions has CMA- and DAPI-bands and this observation indicates that some centromeric region contain AT-rich and CG-rich repeats or Fig. 12. Fluorescent banded chromosomes in Picea close position. Interstitial DAPI-bands appeared at koyamae Hayashi var. acicularis. A: CMA-banding and interstitial region of some chromosomes. B: DAPI-banding. Bar=10μm. In P. maximowiczii var. senanensis four interstitial and one centromeric CMA-bands were observed in the karyotype (Fig. 15A). Lm2 chromosomes had two In P. likiangensis six interstitial CMA-bands and no interstitial CMA-bands on both arms. Two chromosomes centromeric CMA-band were observed (Fig. 13A). Lm2 (Lm3 and Sm1) had interstitial CMA-band and Lm4 chromosomes had two interstitial CMA-bands on both chromosome had centromeric CMA-band. Other eight

FLUORESCENT CHROMOSOME BANDING IN PICEA 23

Fig. 16. Fluorescent banded chromosomes in Picea obovata. A: CMA-banding and B: DAPI-banding. Bar=10μm.

CMA-bands were observed in karyotype (Fig. 17A). Lm3, Lm4, three Lm and Sm1 chromosomes had two CMA-bands at interstitial and centromeric regions. Lm2 Fig. 14. Fluorescent banded chromosomes in Picea and Ssm1 chromosomes had interstitial CMA-band. Other mariana. A: CMA-banding and B: DAPI-banding. Stars chromosomes of Lm1, one Lm, Sm2 and Ssm2 had no shows two types of centromeric CMA-band. Bar=10μm. CMA-band. Faint DAPI-bands appeared at centromeric region of one Lm chromosome and at interstitial region some chromosomes (Fig. 17B).

Fig. 15. Fluorescent banded chromosomes in Picea maximowiczii var. senanensis. A: CMA-banding and B: DAPI-banding. Bar=10μm. chromosomes had no CMA-band. Clear DAPI-band was not observed (Fig. 15B). The fluorescent banding pattern of this species was quite different from other taxa. In P. obovata six interstitial and four centromeric CMA-bands were observed in each karyotype (Fig. 16A). Lm2, Lm3, Lm4, one Lm and Sm1 chromosomes had CMA-bands at the interstitial region. The Lm5 and Sm1 Fig. 17. Fluorescent banded chromosomes in Picea chromosomes had CMA-band at interstitial and omorika. A: CMA-banding and B: DAPI-banding. centromeric regions. Other chromosomes, Lm1, one Lm, Bar=10μm. Sm2 and Ssm2 had no CMA-band. Negative DAPI-bands appeared at the interstitial rereigns of CMA-band and In P. orientalis five interstitial and four centromeric centromeric region of most chromosomes. Thin interstitial CMA-bands appeared in karyotype (Fig. 18A). Lm3, Lm5, DAPI-bands were observed on several chromosomes and one Lm and Ssm1 chromosomes had two CMA-bands at proximal DAPI-band appeared on metacentric B- interstitial and centromeric regions. Lm2 and Lm4 chromosome (Fig. 16B). chromosomes had an interstitial CMA-band. Other In P. omorika eight interstitial and six centromeric chromosomes, Lm1, two Lm and three short chromosomes

24 HIZUME had no CMA-band. Faint DAPI-band appeared at several chromosomes (Fig. 19B). interstitial and centromeric regions of few chromosomes In P. rubens six interstitial and seven centromeric (Fig. 18B). CMA-bands appeared in a karyotype (Fig. 20). Lm2, Lm3, Lm4, one Lm and Ssm1 chromosomes had both centromeric and interstitial CMA-bands. Lm5 chromosomes had an interstitial CMA-band. Lm5 and one Lm chromosomes had a centromeric CMA-band. Lm1, one Lm, Sm2, and Ssm2 chromosomes had no CMA-band.

Fig. 18. Fluorescent banded chromosomes in Picea orientalis. A: CMA-banding and B: DAPI-banding. Bar=10μm.

In P. pungens the CMA-band pattern of this species Fig. 20. Fluorescent CMA-banded chromosomes in was quite different in number of CMA-bands from other Picea rubens. Bar=10μm. species. Fourteen interstitial CMA-bands appeared in a karyotype (Fig. 19A). Lm1, Lm2, one Lm and Sm2 chromosomes had an interstitial CMA-band and the In P. schrenkiana five interstitial and two centromeric CMA-band of Lm1 varied in appearance among plants. CMA-bands were observed in a karyotype (Fig. 21A). Three chromosomes (Lm3, Lm5 and one Lm) had two Lm3 chromosomes had two CMA-bands at the interstitial interstitial CMA-bands on both arms and the Lm5 and one and centromeric regions. Lm2, Lm4, Lm5 and Sm1 Lm chromosomes had a centromeric CMA-band in chromosomes had an interstitial CMA-band. Ssm1 addition to interstitial ones. Lm4, one Lm, Ssm1 had chromosome had a centromeric CMA-band. Other six interstitial and centromeric CMA-bands. Lm2 and one Lm, chromosomes (Lm1, three Lms, Sm2 and Ssm2) had no Sm1 and Ssm2 had an interstitial CMA-band in one arm. CMA-band. Weak DAPI-bands appeared at the interstitial Lm1 of some plants and Sm2 chromosomes had no region of some chromosomes (Fig. 21B). CMA-band. Faint DAPI-bands appeared sometimes in

Fig. 21. Fluorescent banded chromosomes in . A: CMA-banding and B: DAPI-banding. Bar=10μm.

In P. sitchensis six interstitial and five centromeric CMA-bands were observed in a karyotype (Fig. 22A). Lm3 and Lm4 chromosomes had two CMA bands at Fig. 19. Fluorescent banded chromosomes in Picea interstitial and centromeric regions. Lm2, Lm5 and two pungens. A: CMA-banding and B: DAPI-banding. Lm chromosomes had interstitial CMA-band. Lm1, one Bar=10μm. Lm and Ssm1 chromosomes had centromeric CMA-band.

FLUORESCENT CHROMOSOME BANDING IN PICEA 25

Other short chromosomes (Sm1, Sm2 and Ssm2) had no In P. torano four interstitial and three centromeric CMA-band. Faint centromeric and interstitial DAPI-bands CMA-bands were observed (Fig. 24A). Lm4 chromosome appeared on a few chromosomes with negative had CMA-bands in both regions. Lm2, Lm3 and Lm5 had DAPI-bands at CMA-bands (Fig. 22B). Metacentric B- interstitial CMA-band, and one Lm and Ssm1 chromosomes was similar to that of P. glauca in chromosomes had centromeric CMA-band. Lm1 and two CMA-band pattern. Lm and short three chromosomes have not CMA-band. Clear DAPI-band was not observed (Fig. 24B).

Fig. 22. Fluorescent banded chromosomes in Picea sitchensis. A: CMA-banding and B: DAPI-banding. Bar=10μm.

In P. smithiana eight interstitial and two centromeric CMA-bands were observed in a karyotype (Fig. 23A). Fig. 24. Fluorescent banded chromosomes in Picea Interstitial CMA-bands appeared on Lm2-5, two Lm and torano. A: CMA-banding and B: DAPI-banding. Sm1 chromosomes. Lm5 had interstitial CMA-bands on Bar=10μm. both arms. Ssm1 and Sm1 chromosomes have centromeric CMA-bands. Other four chromosomes (Lm1, one Lm, In P. wilsonii five CMA-bands appeared at the Sm2 and Ssm2) had no CMA-band. The Lm5 interstitial regions and one did at a centromeric region (Fig. chromosome having two interstitial CMA-bands in both 25A). Lm2 chromosome had two interstitial CMA-bands arms was specific to this species among Picea species. on the both arms. Lm3, Lm4 and Sm1 chromosomes had Negative DAPI-bands appeared at position of CMA-band an interstitial CMA-band. One Lm chromosome had a and few faint DAPI-bands appeared on certain CMA-band at the proximal region of one arm. Other chromosomes (Fig. 23B). chromosomes had no CMA-band. Weak DAPI-bands appeared at interstitial regions of several chromosomes (Fig. 25B).

Fig. 23. Fluorescent banded chromosomes in Picea smithiana. A: CMA-banding and B: DAPI-banding. Fig. 25. Fluorescent banded chromosomes in Picea Bar=10μm. wilsonii. A: CMA-banding and B: DAPI-banding. Bar=10μm.

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Table 2. Fluorescent CMA-band pattern of chromosomes in 25 taxa of Picea, Pinaceae. No. No. Taxon and distribution Clade** Lm1 Lm2 Lm3 Lm4 Lm5 Lm6-Lm7-Lm8 Ssm1 Sm1 Sm2 Ssm2 B of I of C P. pungens II I/- I2 I+I I+C I+C+I I+C+I I+C I I+C I - I 14 5 P. rubens II - I2+C I+C I+C I I+C C - C I+C - - 6 7 P. mariana II - I2+C I+C I+C I+C I+C C - C I+C - - 6 8 P. glauca II C I2 I+C I+C/I I+C I+C C I C I+C - C 7 8 Bm P. engelmannii II C I2 I+c I+C I+C I+C C - C I+C - C 6 8 Bsm P. sitchensis II C I2 I+C I+C I I C I C - - - 6 5 Bm P. orientalis I - I2 I+C I+C I+C I+C C - I+C - - - 5 4 P. omorika I - I2 I+C I+C I+C I+C I+C - I+C I - - 8 6 P. obovata I - I2 I I I+C I c - C I+C - - 6 4 Bm P. abies I - I2 I I+c I+C I C - C I+C - - 6 5 P. smithiana III - I2 I I I+I I I - C I+C - - 8 2 P. asperata I - I2 I I+C I - - - C I+C - - 5 3 P. jezoensis var. jezoensis I - I2 I I+C I+C I C c/- C I - - 6 4 P. jezoensis var. hondoensis I - I2 I I+C I+C I c - C - - - 5 4 P. glehnii I - I2 I+c I+c I+c I c - C I - - 6 5 Bm P. schrenkiana III - I2 I+C I+c I - - - C I - - 5 2 P. brachytyla* III - I2/I2+I I I+C/I I - - - -/C I - - 4 1 P. alcoquiana I C I2 I+C C I+C C - - C I - - 4 5 P. torano I - I2 I I+C I - C - C - - - 4 3 P. koraiensis I - I2 I I I I - - C -/I+C - - 5 1 P. crassifolia I - I2 I I I - - - C -/I+C - - 4 1/0 Bm P. koyamae var. acicularis I - I2 I I I I - - - i - - 5 0 Bm P. koyamae I - I2 I I I I - - - i - - 5 0 P. maximowiczii var. senanensis I - I2+I I ------I - - 4 0 P. wilsonii III - I2+I I I - l C - - I - - 5 1 P. likiangensis III - I2+I I - I I - - - I - - 6 0 Bsm I: interstitial CMA-band, I2: double interstitial CMA-bands, C: centromeric CMA-band. Small characters are weak CMA-bands. *After Hizume et al (1991) and **clades of Lockwood et al. (2013). Column color of taxon shows distribution; pink is America, beige is Eurasia and Europa and pale green is Asia. CMA-band pattern of chromosome show different color in a cell.

FLUORESCENT CHROMOSOME BANDING IN PICEA 27

CMA-band pattern of each chromosome pair in Picea Sm1 chromosome: Interstitial CMA-bands appeared in In 25 Picea taxa the CMA-band pattern was specific 20 taxa. Out of 20 taxa seven ones had a centromeric for each taxon with little intraspecific variation and varied CMA-band in addition to the interstitial CMA-band. The among taxa (Table 2). Varieties of same species showed other five species had no CMA-band. same or similar CMA-band pattern. Number of interstitial Sm2 chromosome: Any CMA-band did not appear in CMA-bands varied from four to fourteen and of all taxa. centromeric CMA-bands did from zero to eight. In respect Ssm2 chromosome: CMA-band was not observed in to total number of CMA-bands most of American and most taxa. P. pungens had an interstitial CMA-band and, Eurasian species had more than East Asian species. two species, P. engelmannii and P. glauca had a Interstitial CMA-bands varied from four to eight excepting centromeric CMA-band. P. pungens and centromeric CMA-bands showed more variation from zero to eight in number. The CMA-band DISCUSSION pattern was useful to identify homologous chromosome in Fluorescent CMA- and DAPI-band pattern of chromosome each taxon. Among taxa eight chromosome pairs probably complement is useful for identification of homologous homoeologous were identified by chromosome shape and chromosomes and supplies a valuable information on CMA-band through taxa but an identification of other relationships among species in some genera of Pinaceae three Lm chromosomes was difficult. Tendency of such as Abies (Hizume et al. 2016), Larix (Hizume et al. CMA-banding in each identifiable chromosome pair was 1988b, 1994, 1995; Hizume and Tanaka 1990), Pinus described below. (Hizume et al. 1983, 1989b, 1990), Pseudotsuga (Hizume Lm1 chromosome: A pattern of no CMA-band was and Kondo 1992), Cedrus (Dagher-Kharrat et al. 2001) dominant. In three American species (P. sitchensis, P. and Tsuga (Hizume 2015a). In Picea the fluorescent engelmannii, P. glauca) and an East Asian species of P. banding was applied to chromosomes first in P. glehnii alcoquinata had centromeric CMA-band. (Hizume et al. 1988a) and then in several species (Hizume Lm2 chromosome: Characteristic CMA-band pattern et al. 1989a, 1991; Siljak-Yakovlev et al. 2002). CMA- was observed in all species. In two American species (P. bands were clearly observed at the interstitial and/or rubens and P. mariana) centromeric DAPI-band appeared centromeric regions. Clear and faint DAPI-bands were in addition to interstitial CMA-band. The three Asian also observed at the centromeric and interstitial regions in species (P. wilsonii, P. maximowiczii var. senanensis, P. several species, but their appearance seems poorly likiangensis) had CMA-bands at interstitial region of both reproducible. In this time stable CMA-banded karyotypes arms. were analyzed comparatively among Picea taxa. Lm3 chromosome: Half number of species has interstitial CMA-band and the others had Interstitial and Comparative analysis of CMA-banded karyotype centromeric CMA-band. Twelve Eurasian species had only among Picea taxa After an identification of nine an interstitial CMA-band. Five American species and five homoeologous chromosome pairs a CMA-band pattern of Eurasian species had interstitial and centromeric each taxon was compared among taxa. The karyotypes of CMA-bands. P. pungens had two interstitial CMA-bands taxa are arranged based on CMA-band pattern in Table 2. in both arms. An alignment of taxa seems corresponds roughly to their Lm4 chromosome: The dominant CMA band pattern geographic distribution, North America, Eurasia- having centromeric and interstitial CMA-bands appeared Europa and East Asia. American and Arctic Eurasian- in six American species and 11 Eurasian species. Seven European species had many CMA-bands and, interstitial East Asian species had only interstitial CMA-band. P. bands were from 5 to 14 and centromeric bands were from maximowiczii and P. likiangensis had no interstitial 4 to 8. Most East Asian species has less number of CMA-band. interstitial CMA-bands (4-6) and of centromeric Lm5 chromosome: American, Eurasian species had CMA-bands (0-5) than those of American and Eurasian- interstitial and centromeric CMA-bands. Six East Asian European species. In American species P. sitchensis and P. species have only interstitial CMA-band. P. maximowiczii engelmannii had a centromeric CMA-band on Lm1 and P. var. senanensis and P. wilsonii had no CMA-band. rubens and P. mariana had Lm2 chromosomes with the Identification of three Lm chromosomes was possible centromeric and interstitial CMA-bands. P. glauca having in each species but difficult among species. After CMA-banded chromosomes (Lm1, Lm2, Ssm2) might identification of each homoeologous chromosome pair by suggest an intermediate relative among the American other methods the CMA-band pattern of these species groups. P. pungens had abundant the interstitial chromosomes might supply more useful information to CMA-bands (Fig. 19A) than any other taxa. Eurasian relationships among Picea species. species have less centromeric CMA-bands than American Ssm1 chromosome: Centromeric CMA-bands species. European and several Asian species have several appeared in 20 taxa. In three species (P. pungens, P. interstitial CMA-bands like American species. In the orientalis and P. omorika) interstitial CMA-band appeared group of P. orientalis, P. omorika, P. obovata, P. abies and in addition to centromeric one. Six Asian species had no P. smithiana had a similar CMA-band pattern especially in CMA-band. Ssm1 or Sm1. In Asian species some chromosomes having

28 HIZUME specific CMA-band pattern were observed in several some showed fluorescent bands in P. glehnii (Hizume et al. species groups. A unique CMA-band pattern in Lm2 was 1988a, 1989a) and several species of this study (Fig. 6, 12, shared in P. wilsonii, P. maximowiczii var. senanensis and 13, 16, 22, ). Because the B-chromosome dose not pair P. likiangensis. P. glehnii, P. jezoensis, P. torano and P. with A-chromosome, a distribution of each type of brachytyla had similar CMA-banding pattern to each other. B-chromosomes in species will be expected to reveal Chinese species P. koraiensis, P. crassifolia and P. genetic connection among species. asperata also had similar CMA-band pattern. These similarities in the CMA-band pattern might indicate Comparison between fluorescent banding pattern genetic relationships among Picea taxa. and phylogentic relathionship. Several classification Appearance of many interstitial CMA-bands in P. systems of genus Picea were proposed by taxonomists pungens (Fig. 19A) suggests that CMA-bands definitely such as Mayr (1890) and Liu (1982) based on vegetative increase under unknown genetic condition. A tendency of and reproductive structures, and a revision recognized two increase or decrease of CMA-band number in karyotype of sections each with two subsections (Farjon 1990, 2001, Picea species is unresolved. In American species 2010) (see Table 1 in Bouillé et al. 2011). These centromeric CMA-band would increase by amplification taxonomical treatments were somewhat complex and of an unknown GC-rich sequence. A CMA-band pattern difficult to compare with the species alignment based on established once in species and then a CMA-band pattern the CMA-band pattern. Recently several molecular seems not easy to change by deletion, translocation or phylogenetic studies were performed in Picea (Ran et al. transposition of the CMA-band. In one way new 2006, Bouillé et al. 2011, Lockwood et al. 2013). The CMA-band pattern might be constructed between species molecular phylogenetic relationship (Fig. 3 in Lockwood or populations having different band pattern by et al. 2013) seems the most reliable using sequences of hybridization and then introgression. After the three genomes. Picea species was monophyletic and three hybridization between homoeologous chromosomes a clades were recognized; Clade I Asian and three European recombination will also generate new band pattern. species, Clade II have North American species, and Clade In Pinaceae early separate genera (Larix, Pseudotsuga, III had Asian and one American species. An ancestor Cedrus, Pseudolarix, Abies and Tsuga) had small numbers separated Clade I and another clade which later separated of interstitial CMA-bands (Hizume and Tanaka 1990; into Clades II and III. The clades were compared with an Hizume and Kondo 1992; Dagher-Kharrat et al. 2001; alignment of CMA-band pattern (Table 2). In Clade II Hizume 2015a, b; Hizume et al. 2016) and the new genera, some species groups such as P. sitchensis P. engelmannii Pinus and Picea had many interstitial CMA-bands and P. glauca, P. mariana and P. rubens showing similar (Hizume et al. 1983, 1989a, b, 1990). This might suggest CMA-band pattern were put closely in the phylogenetic CMA-bands tend to increase during diversification of . Eurasian species of Clades II and III were mixed in genera. On this point of view East Asia where most CMA-banding alignment. species had small number of interstitial CMA-bands might Paleobotanical studies indicated that Picea would be possible origin site of Picea. differentiate during middle Jurassic (Zackos et al. 2001) In Picea natural and artificial hybridization between and a fossil of P. burtonii was discovered from Early many species had been reported and supplied important Cretaceous (136 Ma) (Klymiuk and Stockey 2012). In the information of genetic relation among species. Natural late Miocene (10-5 Ma) species dispersed over North hybridization and introgression in sympatric area for Hemisphere and species differentiation was presented by examples between P. mariana and P. rubens (Perron and molecule data (Lockwood et al. 2013). The origin site of Bousquet 1997) and P. sitchensis and P. glauca (Szmidt et Picea was proposed East Asia by morphological and al. 1988), and other combination (Sutton et al. 1991; interspecific hybridization (Wright 1955) and North Rajora and Dancik 2000), were reported. The America by molecular phylogeny (Lockwood et al. 2013). introgression increases variability of populations or In any event after an ancestral species spreading and species. Artificial hybridization had been performed in speciation the species would meet by climate changes in various species combination and summarized in Mikkola glacial and interglacial periods, and recurring (1969) and Wright (1955). The Fluorescent banding would hybridization or introgression occurred among species. also as useful tool to identify interspecific hybrids or These phenomena might generate chromosomal and introgression in addition to method of isozyme (Krutovskii genetic complexity of phylogenetic relationships among and Bergmann 1995) and DNA markers (Bobola et al. Picea species. 1996a, b; Perron and Bousquet 1997; Jaramillo-Correa et al., 2004; Du et al. 2009, Chen et al. 2010). We expect to Relationships between fluorescent bands and DNA detect an event of speciation by introgression in Picea by sequences in Picea genome To understand molecular the CMA-banding. nature of CMA- and DAPI-bands a compare between B-chromosomes appeared the most frequently in Picea fluorescent bands with the ISH/FISH signals of certain among genera of Pinaceae (Kruklis 1971; Mori and Fox DNA probes supplied valuable information. In Picea 1972). The B-chromosomes were small metacentric and conventional probes are 45S rDNA, 5S rDNA and Sau3A submetacentric in P. glauca (Teoh and Rees 1977), and tandem repeat. The Sau3A was cloned in genome of P.

FLUORESCENT CHROMOSOME BANDING IN PICEA 29 glauca (Brown et al. 1998) and designed a primer set (Lubaretz et al. 1996; Shibata and Hizume 2008). On three working universally in Picea species (Hizume et al. 1999). centromeric C- or CMA-bands FISH signals of Sau3A In course of genome study in P. abies four repetitive DNA repeat (Shibata and Hizume 2008) and two repetitive clones were selected in a genomic library and used as DNAs (PAF1, IPABCD6) were appeared (Vischi et al. FISH probes (Vischi et al. 2003; Sarri et al. 2008), but did 2003; Sarri et al. 2008). The results indicate the not applied to chromosomes of other species. centromeric regions of two short chromosomes (Ssm1 and Most ISH/FISH signals of these probes appeared Sm1) contained at least three kinds of repeat sequences. corresponding to CMA- and/or DAPI-bands on Out of other nine centromeric C-band three C-bands chromosomes. All interstitial CMA-bands coincided with contained RATRI40 sequence and other five C-band did 45S rDNA signals excepting for a thin CMA-band of Lm2 an unknown repetitive DNA. Ssm1 chromosome having a chromosome (Hizume and Kuzukawa 1995; Hizume et al. DAPI-band at the centromeric region suggests a presence 1999; Siljak-Yakovlev et al. 2002; Shibata and Hizume of AT-rich sequence in addition to two repetitive DNAs 2008). The thin CMA-band locating at the proximal side (PAF1, IPABCD6). A FISH signal of one repetitive DNA of interstitial CMA-band of Lm2 chromosome showed a (2F) appeared at centromeres of all chromosomes as a pair signal of 5S rDNA (Hizume and Kuzukawa 1995; Brown of dots and at most of secondary constrictions (Vischi et al. and Carlson 1997; Hizume et al. 1999; Shibata and 2003). Centromeric FISH signals of Sau3A repeat (Shibata Hizume 2008). The localization of 5S rDNA in Picea and Hizume 2008) is corresponding to just three C-bands. karyotype is different from that of other Pinaceae genera This suggests that some centromeric C-bands might examined (Hizume et al. 1995, 1996, 2002; Dagher- contain a repetitive DNA with neutral base composition Kharrat et al. 2001; Shibata et al. 2004). and contained 2F repeat. The centromeric regions The FISH using Sau3A repeat was applied to containing several repetitive DNAs in some combination chromosomes of P. glauca and P. sitchensis (Brown et al. and composition were different depending on each 1998) and 13 species (Hizume et al. 1999; Shibata and chromosome. The DNA contained in the terminal C-band Hizume 2008), and the signals appeared at the centromeric was not detected by other techniques. FISH results region of some chromosomes in all species examined. The (Siljak-Yakovlev et al. 2002; Vischi et al. 2003; Shibata CMA-band pattern was compared with Sau3A signal and and Hizume 2008) suggest that the interstitial and then the Sau3A signal appeared on some centromeric centromeric C-bands or fluorescent bands have several CMA-bands. In American species (P. rubens, P. mariana, repeat sequences with various compositions. P. engelmannii, P. pungens) have several centromeric DAPI-bands appeared at the interstitial and CMA-bands but out of them three or four centromeric centromeric regions in several species, P. abies, P. CMA-bands correspond to Sau3A signals. This indicates omorika (Siljak-Yakovlev et al. 2002), and P. asperata that some centromeric CMA-bands are composed of (Fig. 3B), P. engelmannii (Fig. 4B), P. glauca (Fig. 6B), P. Sau3A repeat and the other centromeric CMA-bands likiangensis (Fig. 13B) and others like those observed in contained a different repeat sequence. Pinus species (Hizume et al. 1983, 1989b, 1990). P. abies is one of the most investigated species on its Detection of DAPI-bands was somewhat unstable in Picea, genome (Nystedt et al. 2013) and, its chromosomal and then could not be analyzed detail in this paper. The information on conventional karyotype, banding and FISH optimization of DAPI-banding technique for Picea signal pattern are deposited. The various information of chromosomes and a development of FISH AT-rich probe chromosome is inspected by comparing them as following. for DAPI-band are desired to clarify more detailed Typical karyotype has six secondary constrictions at an understanding of chromosome organization and of interstitial region of five long metacentric chromosomes relationship and comparison of chromosome compliment and a short chromosome (Terasmaa 1971, 1975; Hizume or genome in Picea species. 1988). The C-banding, which stains differentially a chromosome segment of highly repetitive DNA, was The CMA-band pattern of chromosomes in Picea applied to haploid endosperm cells. C-bands appeared at species would contain valuable information on the interstitial regions of three chromosomes, the relationship among species. The relationships of species telemetric regions of two chromosomes and the are so complex that more molecular cytogenetic study and centromeric regions of nearly all chromosomes (Kohler et reliable molecular phylogenetical study are needed. In al. 1996). Interstitial C-bands coincided with CMA-bands. evolution a hybridization and polyploidization in Three centromeric C-bands were CMA-bands but other various level seems more an important generator than gene centromeric C-bands did not show fluorescent band. mutation. In plants like Picea having a stable chromosome Application of FISH with various probes revealed karyotype, species differentiation might drive by a chromosomal organization of C-bands and fluorescent combination of chromosome in a complement and/or bands. The 45S rDNA (Lubaretz et al. 1996; Shibata and recombination after interspecific hybridization. More Hizume 2008), PAF1 and IPABCD6 (Vischi et al. 2003; molecular cytogenetic study using universal Picea-specific Sarri et al. 2008) were localized on all secondary probes is critical to understand of chromosomal constrictions (interstitial CMA-bands or C-bands). Thin relationship or phylogeny of Picea species. CMA-band of Lm2 corresponded with 5S rDNA signal

30 HIZUME

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