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Cytogenetic Diversity at Population Level in Iranian Five Species of Genus Cirsium (Asteraceae)

Masoud Sheidaia,*, Reyhaneh haji-Ramezanalia, Simin zanganeha, Maryam nouRoozia and Zahra nooRmohammadib

aFaculty of Biological Sciences, Shahid Beheshti University, Tehran, IRAN; bDepartment of Biology, School of Sciences, Science and Research Branch,

Islamic Azad University, Tehran, IRAN*Corresponding author: msheidai@yahoo.com, msheidai@sbu.ac.ir

(Accepted on May 10, 2014)

About 44 Cirsium Mill. (Asteraceae) species grow in Iran with various geographical populations and great morphological and potential genetic variability. The present study considers cytological features of 23 populations in five Cirsium species comparing their polyploidy level and meiotic behavior. The populations of C. echinus, C. pyramidale, C. congestum and C. spectabile showed 2n = 2x = 34 chromosome number, while C. vulgare had 2n = 4x = 68. Cirsium pyramidale chromosome number is new from Iran. All populations showed mostly bivalents, although few univalents and quadrivalents were also formed. The species and populations differed significantly in their relative chiasma frequency and chromosome pairing. Diploid species C. pyramidale, C. congestum and C. spectabile formed 1–2 quadrivalents due to the occurrence of heterozygote translocations. Meiotic abnormalities including chromosome stickiness, laggards and multipolar cells occurred in some of the species and populations.

Key words: Chromosome pairing, Cirsium, Iran, meiotic abnormalities.

J. Jpn. Bot. 89: 335–345 (2014)

The genus Cirsium Mill. (Asteraceae) contains about 250 perennial, biennial or rarely annual and spiny species (Bureš et al. 2004), which mainly grow in the Northern Hemisphere. These species show wide ranges of ecological adaptations in places they grow and occupy different localities with regard to elevation, temperature and edaphic factors due to their genetic adaptability and plasticity. They are well known for interspecific hybrid formation and wide gene flow, which helps their adaptation to various environmental conditions (Bureš et al. 2004).

About 44 Cirsium species have been reported in Flora Iranica (Rechinger 1979), which have

been classified in five sections. These species grow in different regions of the country and contain several geographical populations with great morphological and genetic variability as also reported in C. arvense studied recently (Sheidai et al. 2012).

Nouroozi et al. (2010), studied cytogenetic diversity in 21 populations of 17 Cirsium species growing in Iran. They reported some new chromosome numbers and showed significant difference in chromosome pairing and chiasma frequency among the species and populations studied. We tried here to continue this study by focusing on the species and geographical populations that were not studied by Nourozzi

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et al. (2010). Therefore, we get a better picture of cytological diversity occurring along with molecular diversity operating in Cirsium populations diversification.

In general we have few cytological reports on the genus Cirsium (Frankton and Moore 1961, Tonian 1981a, 1981b, Dempsey 1994, Ghaffari 1999, Ozcan et al. 2008), and only recently some cytological studies have been published from Iran (Nouroozi et al. 2010, 2011). The present study considers cytological features of 23 populations in five Cirsium species of Iran concerning polyploidy level, chiasma frequency and distribution and chromosome pairing as well as meiotic abnormalities for the first time. Chiasma frequency and distribution are useful data in showing species distinctness

and population diversity as well as species relationship as reported in several plant species (i.e., Sheidai et al. 2009, 2010, 2012).

Materials and MethodsPlant materials

While visiting the natural habitats of Circium species in the country, we collected suitable flower buds from 23 geographical populations of five species (Fig. 1). These species are from three sections namely: sect. Psudoepitrachys Petrak, Cirsium spectabile DC. E. (one population), C. congestum Fisch. & C. A. Mey. (six populations), and C. pyramidale Bornm. (six populations). Sect. Echenais (Cass.) Petrak: C. echinus (M. Bieb.) Sch. Bip. (six populations). Sect. Epitrachys DC.: C. vulgare (Savi) Ten.

Table 1. Locality and voucher specimen of the Iranian Cirsium species studiedSpecies Locality Province Latitude Longitude Height (m) Voucher (HSBU)C. echinus Damavand Hamedan 35°43′04.66″N 52°03′56.16″E 1983 1227C. echinus Heyranpassage Ardebil 38°24′35.25″N 48°35′50.26″E 1600 1228C. echinus Asbdavani Ardebil 37°42′41.65″N 48°56′23.31″E 84 1229C. echinus Ahar East Azarbayejan 38°35′26.81″N 47°03′41.87″E 1782 1226C. echinus Khalkhal Ardebil 37°37′06.39″N 48°31′44.93″E 1803 1225C. echinus Eskanloo East Azarbayejan 39°08′21.59″N 47°04′12.74″E 677 1230C. vulgare Baladeh Mazandaran 36°28′49.98″N 51°56′48.65″E 352 1231C. vulgare Mishoodagh East Azarbayejan 38°19′40.89″N 45°38′22.95″E 2889 1242C. vulgare Klibar-Ahar East Azarbayejan 38°42′10.11″N 47°03′00.27″E 1990 1240C. vulgare Heyranpassage Ardebil 38°24′35.25″N 48°35′50.26″E 1005 1241C. pyramidale Hankaei Kerman 29°32ʹ04.12″N 56°25′34.33″E 2797 1200C. pyramidale Gonbadan Kerman 29°26ʹ35.55″N 56°28′39.91″E 2579 1201C. pyramidale Moosaroeiyeh Kerman 29°16ʹ03.07″N 56°48′04.36″E 2546 1202C. pyramidale Divanbala village Kerman 29°19ʹ07.01″N 56°50′53.93″E 2570 1203C. pyramidale Ramoo valley Kerman 29°18ʹ12.58″N 57°09′13.30″E 2591 1204C. pyramidale Dalfard Kerman 29°14ʹ27.89″N 57°16′11.38″E 2705 1205C. congestum Manjil Gilan 36°49ʹ32.50″N 49°25′19.84″E 259 1206C. congestum Gachsar Alborz 36°08ʹ57.41″N 51°19′10.91″E 2247 1209C. congestum 10 km to Manjil Gilan 36°44ʹ49.89″N 49°25′05.00″E 416 1208C. congestum Zanjan-Tehran Zanjan 36°12ʹ12.32″N 49°11′13.30″E 1665 1210C. congestum Khor village Alborz 36°00ʹ45.19″N 50°43′00.99″E 1540 1211C. congestum Ghoshchi passage West Azarbayejan 38°03ʹ20.06″N 44°50′29.03″E 1801 1212C. spectabile Ghomeyshloo Esfahan 32°53ʹ16.02″N 51°08′53.25″E 2180 1219

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(four populations). Young flower buds were obtained from

10–15 randomly selected plants in each population. Plant specimens were collected from natural habitats of these species distributed in nine different provinces of the country with different latitude, longitude as well as altitude showing variation in environmental and climatic conditions (Table 1). Voucher specimens have been deposited in the Herbarium of Shahid Beheshti University (HSBU).

Cytological studies Meiotic studies were performed on young

flower buds collected using the minimum of 100 metaphase/ diakinesis pollen mother cells (PMCs) and 500 anaphase and telophase cells for data collection. Staining was done by acetocarmine (2%) using the squash method as reported before (Sheidai et al. 2012). Pollen satiability as a measure of fertility was determined by staining minimum 1000 pollen grains with 2 % acetocarmine: 50% glycerin (1:1) for about ½ hr. Round, complete pollen grains which were stained were taken as fertile, while incomplete, shrunken pollens with no stain were considered infertile (Sheidai et al.

2012). Meiotic characteristics including chiasma frequency and distribution, chromosome pairing as well as meiotic abnormalities (laggard chromosomes, chromosome stickiness, micronucleus formation, etc.) were determined in the populations studied.

Data analysesIn order to determine significant difference

in meiotic characteristics among the species, ANOVA (analysis of variance) test was performed. Single linkage clustering was also performed on standardized cytological data (mean = 0, variance = 1) to show the population’s cytological diversity in each species (Podani 2000). Pearson coefficient of correlation was determined among cytological characters. χ2 test was performed to study significant difference in meiotic abnormalities among the populations. SPSS ver. 9 (1998) and NTSYS ver. 2. (1998) software was used for statistical analyses.

ResultsCytological features of the species and

populations studied are presented in Table 1 and Figs. 2–4. The populations of Cirsium echinus, C. pyramidale, C. congestum and C. spectabile showed 2n = 2x = 34 chromosome number, while C. vulgare had 2n = 4x = 68 (Fig. 2), supporting the results presented by Nouroozi et al. (2010). The chromosome number for C. pyramidale is new to Iran.

All populations formed mostly bivalents, although some amount of univalents and quadrivalents were also formed (Table 1). Since the species differ in polyploidy level, relative meiotic values (cytological data divided by chromosome number) was obtained to compare different populations and species studied (Table 1).

The highest value of total chiasmata per bivalent occurred in the Heyran passage population of C. echinus (1.68) followed by the Eskanloo populations of C. echinus (1.63). The lowest value of the same parameter occurred in

Fig. 1. Geographical distribution of the Cirsium species studied. c. C. congestum. e. C. echinus. p. C. pyramidale. s. C. spectabile. v. C. vulgare.

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Table 2. Actual and relative meiotic characters in the Iranian Cirsium species studied Species 2n TX IX TOX I RD RB IV TXN IXN TOXN IN RDN RBN IVN

C. echinus 34 24.64 0.00 24.64 0.29 8.79 7.93 0.00 1.45 0.00 1.45 0.02 0.52 0.47 0.00

C. echinus 34 22.85 0.04 22.81 1.15 8.58 7.15 0.00 1.68 0.00 1.68 0.07 0.50 0.42 0.00

C. echinus 34 24.47 0.06 24.41 0.82 8.00 8.29 0.00 1.44 0.00 1.44 0.05 0.47 0.49 0.00

C. echinus 34 25.29 0.00 25.29 0.41 7.88 9.29 0.00 1.49 0.00 1.49 0.02 0.46 0.55 0.00

C. echinus 34 23.20 0.00 23.20 0.53 9.73 6.73 0.00 1.36 0.00 1.36 0.03 0.57 0.40 0.00

C. echinus 34 28.00 0.00 28.00 0.20 5.60 11.20 0.00 1.65 0.00 1.65 0.01 0.33 0.66 0.00

C. vulgare 68 55.93 0.43 55.50 0.79 10.07 23.14 0.00 1.64 0.01 1.63 0.02 0.30 0.68 0.00

C. vulgare 68 51.85 0.23 51.62 0.15 15.69 18.15 0.00 1.52 0.01 1.52 0.00 0.46 0.53 0.00

C. vulgare 68 55.65 0.20 55.45 1.20 10.00 22.70 0.20 1.64 0.01 1.63 0.04 0.29 0.67 0.01

C. vulgare 68 52.44 0.19 52.25 1.75 12.13 20.00 0.13 1.54 0.01 1.54 0.05 0.36 0.59 0.00

C. pyramidale 34 22.08 0.24 21.84 2.40 9.52 4.84 0.72 1.30 0.01 1.28 0.14 0.56 0.28 0.04

C. pyramidale 34 20.07 0.07 20.00 2.67 11.3 3.90 0.23 1.18 0.00 1.18 0.16 0.66 0.23 0.01

C. pyramidale 34 19.94 0.00 19.94 3.00 11.06 4.31 0.06 1.34 0.00 1.34 0.18 0.65 0.25 0.00

C. pyramidale 34 24.37 0.00 24.37 1.16 8.47 7.00 0.47 1.43 0.00 1.43 0.07 0.50 0.41 0.03

C. pyramidale 34 34.34 0.00 34.34 4.58 12.34 2.42 0.04 1.01 0.00 1.01 0.27 0.72 0.14 0.00

C. pyramidale 34 19.56 0.03 19.54 2.97 11.44 3.51 0.28 1.15 0.00 1.15 0.34 0.67 0.21 0.02

C. congestum 34 22.48 0.22 22.26 2.52 9.19 6.04 0.26 1.32 0.01 1.31 0.15 0.54 0.36 0.02

C. congestum 34 21.71 0.19 21.52 0.97 11.26 5.03 0.19 1.28 0.01 1.27 0.06 0.66 0.30 0.01

C. congestum 34 18.83 0.00 18.83 2.27 12.77 2.60 0.20 1.11 0.00 1.11 0.13 0.75 0.15 0.01

C.congestum 34 20.19 0.00 20.19 1.88 11.88 4.00 0.06 1.19 0.00 1.19 0.11 0.70 0.24 0.00

C. congestum 34 23.10 0.10 23.00 0.40 10.50 5.90 0.20 1.36 0.01 1.35 0.02 0.62 0.35 0.01

C. congestum 34 19.50 0.00 19.50 0.80 13.70 2.90 0.00 1.15 0.00 1.15 0.05 0.81 0.34 0.00C. spectabile 34 19.68 0.12 19.56 0.44 13.48 3.00 0.04 1.16 0.01 1.15 0.03 0.79 0.18 0.00Abbreviations: TX. Terminal chiasmata. IX. Intercalary chiasmata. TOX. Total chiasmata. RB. Ring bivalents. RD. Rod bivalents. I. Univalents. IV. Quadrivalents. TXN. Terminal chiasmata/bivalent. IXN. Intercalary chiasmata/bivalent. TOXN. Total chiasmata/bivalent. RBN. Ring bivalents/cell. RDN. Rod bivalents/cell. In. Univalents/cell. IVN. Quadrivalents/cell.

the Manjil population of C. congestum (1.11). A very low value of intercalary chiasmata

occurred in species studied with the highest value (0.43) occurring in the Baladeh population of C. vulgare. However, the values of intercalary chiasmata per bivalent ranged from 0.00–0.01 in the species studied.

The highest value of ring bivalents per cell occurred in the Baladeh population of C. vulgare (0.68), followed by the Klibar-Ahar population of C. vulgare (0.67). The lowest value of the same parameter occurred in the Ramo valley population of C. pyramidale (0.14), followed by the Manjil population of C. congestum (0.15).

One to two quadrivalents (Fig. 1, A) occurred in some of the species studied including diploid species of C. pyramidale, C. congestum and C. spectabile, which were expected to form only bivalents.

Pearson correlation showed significant positive correlation between chromosome number and relative values of total, terminal and intercalary chiasmata, as well as the mean value of ring bivalents per cell (r>0.60, p<0.01). It was negatively correlated with the mean intercalary chiasmata per bivalent and the mean value of rod bivalents per cell (r = −37 and r = −0.66 respectively).

October 2014 TheJournal of Japanese Botany Vol. 89 No. 5 339

Fig. 2. Representative meiotic cells showing chromosome pairing in the Cirsium species and populations studied. A. Metaphase-I cell in Ghomayshloo population of C. spectabile showing bivalents and quadrivalents (arrows). B–D. Metaphase-I cell in Moosaroeiyeh, Gonbadan and Khor village populations of C. pyramidale showing bivalent and univalent formation. E. Metaphase-I cell in Gachsar population of C. congestum showing bivalents. F. Metaphase-I cells in Heyran passage population of C. vulgare showing bivalents. G. Metaphase-I cells in Asbdavani population of C. echinus showing chromosome pairing. Scale bar = 10 µm.

Single linkage clustering performed in each species showed meiotic variations among the populations of the species studied (Figs. 2–4). For example, among the C. echinus populations, the Skanloo populations differed much from the other populations and joined them with distance. Two populations of Mishoodagh and Damavand showed more similarity in their meiotic features, while the Heyran population joined them with

some distance (Fig. 4, A). In C. congestum, the Zanjan-Tehran population differed from the other populations and was placed far from the other populations (Fig. 4, B). Among C. congestum populations, Manjil and Ghoshchi showed high meiotic similarities. Similarly, in C. vulgare, the Marand and Heyran populations differed from the others (Fig. 4, C), while in C. pyramidale, the Hankaei and Ramo populations

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differed from the other populations and were placed far from them (Fig. 4, D).

Various meiotic abnormalities including, laggard chromosomes, stickiness, micronucleus formation and unorganized chromosomes, were observed in almost all populations studied. Chromosomes stickiness occurred in both metaphase of meiosis I and II, between two or more bivalents/ forming a complete clump in some of the cases. The highest percentage of metaphase I cells showing chromosome stickiness was observed in the Divanbala village population of C. pyramidale (38.30%), followed by the Delfard passage population (24.00%). The Ramo valley population of C. pyramidale showed the highest percentage of complete metaphase-I stickiness/clumping (33.3%).

Similarly the Manjil population in C.

congestum and the Asbdavani population in C. echinus showed the highest percentage of metaphase II cells showing chromosome stickiness (16.60%), followed by the Ahar population in C. vulgare (13.00%).

Chromosome stickiness during anaphase I and II which lead to the formation of anaphase bridges were also observed. The number and thickness of these bridges varied among different meiocytes. The highest percentage of anaphase-I cells showing laggard chromosomes occurred in Ahar population (19.00%) and Baladeh populations (16%) of C. vulgare. These laggards may be the reason for micronuclei formation in telophase I and II. χ2 test performed showed significant difference (p<0.05) for meiotic abnormalities among the populations studied.

ANOVA test followed by LSD test

Fig. 3. Representative meiotic cells showing cytological abnormalities in the Cirsium species and populations studied. A. Telophase-I cells in Ghomayshloo population of C. spectabile showing micronucleus. B. Anaphase-II cell in Ramoo valley population of C. pyramidale showing laggard chromosome. C. Metaphase-I cells in Asbdavani population of C. echinus showing chromosome clumping. D. Anaphase-I cell showing chromosome stickiness in Heyran passage population of C. vulgare. Scale bar = 10 µm.

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A: Cirsium echinus

B: Cirsium congestum

C: Cirsium vulgare

Fig. 4. Single linkage dendrogram showing the meiotic diversity in the four Cirsium species populations.

D: Cirsium pyramidale

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performed among the species showed significant difference for chiasma frequency and chromosome association. For example, C. vulgare showed significantly higher mean values for relative total and intercalary chiasmata, while C. pyramidale showed significant higher mean value for quadrivalent formation.

Pearson coefficient of correlation determined among the meiotic characters showed significant positive correlation between the chromosome number and relative total and terminal chiasmata per bivalent as well as with the mean number of ring bivalents per cell (r>0.60, p<0.01). Terminal chiasma per bivalent was negatively correlated to mean number of univalents and rod bivalents per cell (r = − 0.66 and 0.94, p<0.01, respectively).

Pollen fertility test showed that the species

and the populations studied had above 90% pollen stainability. The low degree of infertility observed may be due to the effects of cytological abnormalities observed.

DiscussionThe species and the populations studied

showed 2n = 2x = 34 and 2n = 4x = 68 chromosome numbers indicating that polyploidy is a major cytological and evolutionary mechanism in the Cirsium genus speciation. It has been suggested that 2n = 34 based on x = 17 is the primitive and ancestral chromosome number in the genus Cirsium (Moore and Frankton 1963, Dabeydeen 1980). The most frequent chromosome number within the genus Cirsium is diploid (2n = 34), which is reported for approximately 69% of the species. However, tetraploid (2n = 68) is also relatively common, occurring approximately in 10% of Cirsium species. The less frequent chromosome numbers reported are 2n = 30 and 2n = 32 (in 5% of the species; Bureš et al. 2004).

Among populations of each species studied, no change in chromosome number was encountered which may indicate that the polyploidy level in each species is fixed although these populations grow in different geographical regions facing various environmental conditions. However, the populations in each species differ greatly in chiasma frequency and distribution as well as chromosome pairing which indicate a change in meiotic control over chromosome pairing and chiasma formation during species diversification. Variation in chiasma frequency and localization is genetically controlled (Quicke 1993) and has been reported in several plant species as well as in crop plant varieties (Rees and Dale 1974, Rees and Jones 1977).

Diploid species, C. pyramidale , C. congestum and C. spectabile,are expected to form only bivalents but showed the occurrence of quadrivalents as a result of heterozygote translocation. Such heterozygote translocations may lead to the formation of new linkage

Table 3. ANOVA test of meiotic characters in the Cirsium species studied

Sum of squares df Mean

square F Sig.

TXN Between groups 0.56 4 0.14 11.90 0.01Within groups 0.22 19 0.01Total 0.79 23

IXN Between groups 0.00 4 0.00 1.73 0.19Within groups 0.00 19 0.00Total 0.00 23

TOXN Between groups 0.56 4 0.14 12.25 0.01Within groups 0.22 19 0.01Total 0.78 23

IN Between groups 0.07 4 0.02 9.16 0.01Within groups 0.04 19 0.00Total 0.11 23

RDN Between groups 0.43 4 0.11 15.85 0.01Within groups 0.13 19 0.01Total 0.56 23

RBN Between groups 0.57 4 0.14 19.40 0.01Within groups 0.14 19 0.01Total 0.71 23

IVN Between groups 0.00 4 0.00 4.00 0.02Within groups 0.00 19 0.00Total 0.00 23

Abbreviations: TXN. Terminal chiasmata/bivalent. IXN. Intercalary chiasmata/bivalent. TOXN. Total chiasmata/bivalent. RBN. Ring bivalents/cell. RDN. Rod bivalents/cell. IN. Univalents/cell. IVN. Quadrivalents/cell.

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groups. A similar case has been reported in different populations of C. arvense (Sheidai et al. 2012). Therefore, both mechanisms of a change in chiasma frequency and the occurrence of heterozygote translocations in the populations with the same chromosome number may be a means for generating new forms of recombination influencing the variability within natural populations in an adaptive way (Rees and Dale 1974). The present findings are in agreement with the reports of Nouroozi et al. (2010) on the other studied Cirsium species in Iran.

Cirsium vulgare which is a tetraploid species showed diplontic behavior and did not form any quadrivalent. This has been also reported before in tetraploid species of Cirsium lappaceum (Nouroozi et al. 2010). This type of meiotic behavior either is due to allotetraploid nature of these species or meiotic control over chiasma formation. Therefore, the present study shows cytogenetic diversity available in four species of C. pyramidale, C. congestum, C. echinus and C. vulgare populations that is possibly related to their geographical distribution and adaptive nature of these changes. This suggestion is also

supported by our molecular study performed in the same species and populations (Sheidai et al. 2013). The species showed extensive inter-population molecular diversity (ISSR, Inter Simple Sequence Repeats). Therefore, genetic diversity at both cytogenetic and molecular levels, are operative in Cirsium species diversity and population divergence. However, whether genetic diversity observed bring about morphological changes in the populations studied and if we can recognize any intraspecific taxa, need further morphological investigation which is under way.

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Fig. 5. UPGMA dendrogram of relative meiotic data in the Cirsium species studied. c. C. congestum. e. C. echinus. p. C. pyramidale. s. C. spectabile. v. C. vulgare.

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Fig. 6. PCA biplot of cytogenetic characters in the Cirsium species studied.

Fig. 7. CCA plot of cytogenetic data in the Cirsium species studied.

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M. Sheidaia, R. Haji-Ramezanalia, S. Zanganeha, M. Nouroozia, Z. Noormohammadib：イラン産アザミ属植物（キク科）5種の集団レベルでの細胞遺伝学的多様性　イランにはアザミ属（キク科）に約 44種があり，本研究ではそのうちの 5種 Cirsium echinus，C. pyramidale，C. congestum，C. spectabile，C. vulgareの 23集団で倍数性と減数分裂を調べた．その結果，C. echinus，C. pyramidale，C. congestum，C. spectabileの 4種は 2n = 2x = 34の二倍体種で，C. vulgare は 2n = 4x = 68の四倍体種であることが分かった．このうち，C. pyramidaleの染色体数 2 = 34はイランで初めての報告である．調べた 5種の大部分の集団で二価染色体が見られたが，一価染色体と四価染色体も観察さ

れた．キアズマ頻度や染色体の対合においては種ごとの有意差が観察された．二倍体種の C. pyramidale，C. congestum，C. spectabileでは異型接合のために 1〜 2個の四価染色体が観察された．染色体の粘着性，ハシゴ形成，多極性細胞などの減数分裂における異常性はいくつかの種において観察された．

（aイラン・Shahid Beheshti University，Faculty of Biological Sciences，

bイラン・Islamic Azad University，Department of Biology）